Polypeptides and Antibodies

ABSTRACT

Polypeptides are provided. Antibodies or antigen binding domains are provided which bind such polypeptides. Also provided are methods of obtaining an antibody that binds tumor necrosis factor (TNF)-related apoptosis-inducing ligand (“TRAIL”) Receptor-2 (TR-2) comprising administering at least one of such polypeptides to an animal and obtaining an antibody that binds TR-2 from the animal. Antibodies reactive with TR-2 are provided. Also provided are cells producing antibodies reactive with TR-2, pharmaceutical compositions comprising antibodies reactive with TR-2, methods using antibodies reactive with TR-2, and kits comprising antibodies reactive with TR-2. Also provided are methods of decreasing or preventing binding of an antibody to TR-2 by administering such a polypeptide.

This application claims the benefit of U.S. Provisional Application No.60/713,433, filed Aug. 31, 2005, and U.S. Provisional Application No.60/713,478, filed Aug. 31, 2005. U.S. Provisional Application Nos.60/713,433 and 60/713,478 are incorporated by reference herein in theirentirety for any purpose.

FIELD

Polypeptides are provided. Antibodies or antigen binding domains areprovided which bind such polypeptides. Also provided are methods ofobtaining an antibody that binds tumor necrosis factor (TNF)-relatedapoptosis-inducing ligand (“TRAIL”) Receptor-2 (TR-2) comprisingadministering at least one of such polypeptides to an animal andobtaining an antibody that binds TR-2 from the animal. Antibodiesreactive with TR-2 are provided. Also provided are cells producingantibodies reactive with TR-2, pharmaceutical compositions comprisingantibodies reactive with TR-2, methods using antibodies reactive withTR-2, and kits comprising antibodies reactive with TR-2. Also providedare methods of decreasing or preventing binding of an antibody to TR-2by administering such a polypeptide.

BACKGROUND

The interaction between TR-2 and its ligand, TRAIL, plays a role in theinduction of apoptosis (see, for example, Almasan et al., Cytokine &Growth Factor Reviews 14: 337-348 (2003)). TRAIL, also known as Apo2ligand, is a homomeric ligand that interacts with four members of theTNF-receptor superfamily (TRAIL receptors (“TR”) 1 to 4), as well aswith the related, soluble, osteoprotegerin (“OPG”) receptor. Binding ofTRAIL to TR-1 or TR-2 at the surface of a cell triggers apoptosis ofthat cell. After initial binding of TRAIL to TR-1 or TR-2, intracellularproteins are recruited to the intracellular death domain of thereceptor, forming a signaling complex. Certain intracellular caspasesare recruited to the complex, where they autoactivate and in turnactivate additional caspases and the intracellular apoptosis cascade.TR-3 and TR-4 and OPG lack the intracellular domain responsible fortransmitting the apoptosis signal. Thus, binding of TRAIL to TR-3, TR-4,or OPG does not trigger apoptosis. TR-3 and TR-4 are also referred to as“decoy” receptors, and their overexpression has been shown to protectcells from apoptotic induction by TRAIL. TR-2 is expressed in a varietyof cells, including liver, brain, breast, kidney, colon, lung, spleen,thymus, peripheral blood lymphocytes, prostate, testis, ovary, uterus,and various tissues along the gastro-intestinal tract. (See, forexample, Walczak et al., EMBO J. 16: 5386-5397 (1997); Spierings et al.,J. Histochem. Cytochem. 52: 821-831 (2004)). Though TRAIL and TRAILreceptors are widely expressed, they are most active in inducingapoptosis in transformed cells. (See, for example, Daigle et al., SwissMed. Wkly. 131: 231-237 (2001)).

SUMMARY

In certain embodiments, an isolated polypeptide is provided comprisingat least one complementarity determining region (CDR) selected fromCDR1a, CDR2a, and CDR3a:

-   -   wherein CDR1a comprises the amino acid sequence a b c d e f g h        i j k l, wherein amino acid a is glycine, amino acid b is        selected from glycine, tyrosine, or phenylalanine; amino acid c        is selected from serine or threonine; amino acid d is selected        from isoleucine or phenylalanine; amino acid e is selected from        serine, threonine, or asparagine; amino acid f is selected from        serine, aspartic acid, tyrosine, asparagine, threonine, or        glycine; amino acid g is selected from glycine, aspartic acid,        or tyrosine; amino acid h is selected from glycine, aspartic        acid, tyrosine, asparagine, or serine; amino acid i is selected        from tyrosine, isoleucine, histidine, methionine, or tryptophan;        amino acid j is selected from asparagine, tyrosine, histidine,        serine, or phenylalanine; amino acid k is tryptophan or is not        present; and amino acid l is serine or is not present;    -   wherein CDR2a comprises the amino acid sequence m n o p q r t u        v w x y z a′ b′ c′, wherein amino acid m is selected from        tryptophan, tyrosine, histidine, valine, glutamic acid, or        serine; amino acid n is selected from methionine or isoleucine;        amino acid o is selected from asparagine, tyrosine, serine,        tryptophan, or histidine; amino acid p is selected from proline,        tyrosine, serine, arginine, histidine, or asparagine; amino acid        q is selected from asparagine, serine, or aspartic acid; amino        acid r is selected from serine or glycine; amino acid s is        selected from aspartic acid, serine, threonine, or arginine;        amino acid t is selected from asparagine, threonine, alanine,        isoleucine, or tyrosine; amino acid u is selected from        threonine, tyrosine, leucine, lysine, asparagine, or isoleucine;        amino acid v is selected from glycine, tyrosine, aspartic acid,        or cysteine; amino acid w is selected from tyrosine or        asparagine; amino acid x is selected from alanine or proline;        amino acid y is selected from glutamine, serine, or aspartic        acid; amino acid z is selected from lysine, leucine, or serine;        amino acid a′ is selected from phenylalanine, lysine, or valine;        amino acid b′ is selected from glutamine, serine, or lysine; and        amino acid c′ is glycine or is not present; wherein CDR3a        comprises the amino acid sequence d′ e′ f′ g′ h′ i′ j′ k′ l′ m′        n′ o′ p′ q′ r′ s′ t′ u′ v′ w′, wherein amino acid d′ is selected        from tryptophan, aspartic acid, glycine, serine, or glutamic        acid; amino acid e′ is selected from asparagine, aspartic acid,        glycine, arginine, serine, valine, or leucine; amino acid f′ is        selected from histidine, serine, alanine, tyrosine, proline,        asparagine, glycine or threonine; amino acid g′ is selected from        tyrosine, serine, alanine, arginine, tryptophan, glycine or        valine; amino acid h′ is selected from glycine, alanine, serine,        asparagine, methionine, tyrosine, tryptophan, cysteine, or        aspartic acid; amino acid i′ is selected from serine,        tryptophan, glycine, phenylalanine, aspartic acid, tyrosine, or        threonine; amino acid j′ is selected from glycine, threonine,        serine, leucine, valine, asparagine, tryptophan, or tyrosine;        amino acid k′ is selected from serine, phenylalanine, aspartic        acid, tryptophan, glycine, or tyrosine, or is not present; amino        acid l′ is selected from histidine, aspartic acid, alanine,        tryptophan, tyrosine, serine, phenylalanine, valine, or glycine,        or is not present; amino acid m′ is selected from phenylalanine,        tyrosine, glutamic acid, proline, aspartic acid, cysteine,        isoleucine, or methionine, or is not present; amino acid n′ is        selected from aspartic acid, phenylalanine, alanine, leucine, or        serine, or is not present; amino acid o′ is selected from        tyrosine, leucine, aspartic acid, phenylalanine, proline, or        valine, or is not present; amino acid p′ is selected from        leucine, aspartic acid, or tyrosine, or is not present; amino        acid q′ is selected from serine or tyrosine, or is not present;        amino acid r′ is tyrosine or is not present; amino acid s′ is        selected from glycine or tyrosine, or is not present; amino acid        t′ is selected from glycine or methionine, or is not present;        amino acid u′ is selected from methionine or aspartic acid, or        is not present; amino acid v′ is selected from aspartic acid or        valine, or is not present; and amino acid w′ is valine or is not        present; and        wherein the polypeptide, in association with an antibody light        chain, binds TRAIL receptor-2 (TR-2).

In certain embodiments, an isolated polypeptide is provided comprisingat least one complementarity determining region (CDR) selected from:

amino acids 26 to 35 of SEQ ID NO: 2;

amino acids 50 to 66 of SEQ ID NO: 2;

amino acids 99 to 110 of SEQ ID NO: 2;

amino acids 26 to 37 of SEQ ID NO: 4;

amino acids 52 to 67 of SEQ ID NO: 4;

amino acids 100 to 109 of SEQ ID NO: 4;

amino acids 26 to 37 of SEQ ID NO: 6;

amino acids 52 to 67 of SEQ ID NO: 6;

amino acids 100 to 109 of SEQ ID NO: 6;

amino acids 26 to 37 of SEQ ID NO: 8;

amino acids 52 to 67 of SEQ ID NO: 8;

amino acids 100 to 109 of SEQ ID NO: 8;

amino acids 26 to 35 of SEQ ID NO: 10;

amino acids 50 to 66 of SEQ ID NO: 10;

amino acids 99 to 110 of SEQ ID NO: 10;

amino acids 26 to 35 of SEQ ID NO: 12;

amino acids 50 to 66 of SEQ ID NO: 12;

amino acids 99 to 111 of SEQ ID NO: 12;

amino acids 26 to 35 of SEQ ID NO: 14;

amino acids 50 to 65 of SEQ ID NO: 14;

amino acids 98 to 111 of SEQ ID NO: 14;

amino acids 26 to 37 of SEQ ID NO: 16;

amino acids 52 to 67 of SEQ ID NO: 16;

amino acids 100 to 109 of SEQ ID NO: 16;

amino acids 26 to 35 of SEQ ID NO: 18;

amino acids 50 to 66 of SEQ ID NO: 18;

amino acids 99 to 105 of SEQ ID NO: 18;

amino acids 26 to 35 of SEQ ID NO: 20;

amino acids 50 to 66 of SEQ ID NO: 20;

amino acids 99 to 118 of SEQ ID NO: 20;

amino acids 26 to 35 of SEQ ID NO: 22;

amino acids 50 to 66 of SEQ ID NO: 22;

amino acids 99 to 118 of SEQ ID NO: 22;

amino acids 26 to 35 of SEQ ID NO: 24;

amino acids 50 to 65 of SEQ ID NO: 24;

amino acids 98 to 108 of SEQ ID NO: 24;

amino acids 26 to 35 of SEQ ID NO: 26;

amino acids 50 to 66 of SEQ ID NO: 26;

amino acids 99 to 110 of SEQ ID NO: 26;

amino acids 26 to 35 of SEQ ID NO: 28;

amino acids 50 to 66 of SEQ ID NO: 28;

amino acids 99 to 117 of SEQ ID NO: 28;

amino acids 26 to 37 of SEQ ID NO: 30;

amino acids 52 to 67 of SEQ ID NO: 30;

amino acids 100 to 111 of SEQ ID NO: 30;

amino acids 26 to 37 of SEQ ID NO: 32;

amino acids 52 to 67 of SEQ ID NO: 32;

amino acids 100 to 111 of SEQ ID NO: 32;

amino acids 26 to 37 of SEQ ID NO: 34;

amino acids 52 to 67 of SEQ ID NO: 34; and

amino acids 100 to 111 of SEQ ID NO: 34;

wherein the polypeptide, in association with an antibody light chain,binds TR-2.

In certain embodiments, an isolated polypeptide is provided comprisingat least one complementarity determining region (CDR) selected fromCDR1b, CDR2b, and CDR3b:

-   -   wherein CDR1b comprises the amino acid sequence a1 b1 c1 d1 e1        f1 g1 h1 i1 j1 k1 l1 m1 n1 o1 p1 q1, wherein amino acid a1 is        selected from arginine or lysine; amino acid b1 is selected from        threonine, alanine, or serine; amino acid c1 is serine; amino        acid d1 is glutamine; amino acid e1 is selected from serine or        glycine; amino acid f1 is selected from isoleucine, leucine, or        valine; amino acid g1 is selected from serine, leucine, or        arginine; amino acid h1 is selected from threonine, serine,        isoleucine, asparagine, arginine, histidine, or tyrosine; amino        acid i1 is selected from tyrosine, arginine, tryptophan,        aspartic acid, or serine; j1 is selected from leucine,        isoleucine, asparagine, tyrosine, or serine; amino acid k1 is        selected from asparagine, glycine, valine, alanine, or leucine;        amino acid l1 is selected from tyrosine, alanine, or asparagine,        or is not present; amino acid m1 is selected from asparagine or        lysine, or is not present; amino acid n1 is selected from        tyrosine, asparagine, or isoleucine, or is not present; amino        acid o1 is selected from leucine or tyrosine, or is not present;        amino acid p1 is selected from aspartic acid or leucine, or is        not present; and amino acid q1 is selected from valine, alanine,        or threonine, or is not present;    -   wherein CDR2b comprises the amino acid sequence r1 s1 t1 u1 v1        w1 x1, wherein amino acid r1 is selected from alanine, aspartic        acid, leucine, tryptophan, glycine, or valine; amino acid s1 is        selected from threonine, valine, glycine, or alanine; amino acid        t1 is serine; amino acid u1 is selected from serine, asparagine,        or threonine; amino acid v1 is selected from leucine,        phenylalanine, or arginine; amino acid w1 is selected from        glutamine, alanine, or glutamic acid; and amino acid x1 is        selected from serine, arginine, or threonine;    -   wherein CDR3b comprises the amino acid sequence y1 z1 a1′ b1′        c1′ d1′ e1′ f1′ g1′, wherein amino acid y1 is selected from        glutamine, methionine, leucine, or histidine; amino acid z1 is        selected from glutamine or lysine; amino acid a1′ is selected        from serine, threonine, alanine, histidine, tyrosine, or        phenylalanine; amino acid b1′ is selected from tyrosine,        leucine, asparagine, or glycine; amino acid c1′ is selected from        serine, glutamine, isoleucine, or lysine; amino acid d1′ is        selected from threonine, phenylalanine, tyrosine, alanine, or        serine; amino acid e1′ is proline; amino acid f1′ is selected        from leucine, phenylalanine, tryptophan, serine, or arginine;        and amino acid g1′ is selected from threonine or serine; and        wherein the polypeptide, in association with an antibody heavy        chain, binds TR-2.

In certain embodiments, an isolated polypeptide is provided comprisingat least one complementarity determining region (CDR) selected from:

amino acids 24 to 34 of SEQ ID NO: 36;

amino acids 50 to 56 of SEQ ID NO: 36;

amino acids 89 to 97 of SEQ ID NO: 36;

amino acids 24 to 34 of SEQ ID NO: 38;

amino acids 50 to 56 of SEQ ID NO: 38;

amino acids 89 to 97 of SEQ ID NO: 38;

amino acids 24 to 34 of SEQ ID NO: 40;

amino acids 50 to 56 of SEQ ID NO: 40;

amino acids 89 to 97 of SEQ ID NO: 40;

amino acids 24 to 34 of SEQ ID NO: 42;

amino acids 50 to 56 of SEQ ID NO: 42;

amino acids 89 to 97 of SEQ ID NO: 42;

amino acids 24 to 34 of SEQ ID NO: 44;

amino acids 50 to 56 of SEQ ID NO: 44;

amino acids 89 to 97 of SEQ ID NO: 44;

amino acids 24 to 34 of SEQ ID NO: 46;

amino acids 50 to 56 of SEQ ID NO: 46;

amino acids 89 to 97 of SEQ ID NO: 46;

amino acids 24 to 40 of SEQ ID NO: 48;

amino acids 56 to 62 of SEQ ID NO: 48;

amino acids 95 to 103 of SEQ ID NO: 48;

amino acids 24 to 39 of SEQ ID NO: 50;

amino acids 55 to 61 of SEQ ID NO: 50;

amino acids 94 to 102 of SEQ ID NO: 50;

amino acids 24 to 40 of SEQ ID NO: 52;

amino acids 56 to 62 of SEQ ID NO: 52;

amino acids 95 to 103 of SEQ ID NO: 52;

amino acids 24 to 34 of SEQ ID NO: 54;

amino acids 50 to 56 of SEQ ID NO: 54;

amino acids 89 to 97 of SEQ ID NO: 54;

amino acids 24 to 34 of SEQ ID NO: 56,

amino acids 50 to 56 of SEQ ID NO: 56;

amino acids 89 to 97 of SEQ ID NO: 56;

amino acids 24 to 40 of SEQ ID NO: 58;

amino acids 56 to 62 of SEQ ID NO: 58;

amino acids 95 to 103 of SEQ ID NO: 58;

amino acids 24 to 34 of SEQ ID NO: 60;

amino acids 50 to 56 of SEQ ID NO: 60;

amino acids 89 to 97 of SEQ ID NO: 60;

amino acids 24 to 34 of SEQ ID NO: 62;

amino acids 50 to 56 of SEQ ID NO: 62;

amino acids 89 to 97 of SEQ ID NO: 62;

amino acids 24 to 35 of SEQ ID NO: 64;

amino acids 51 to 57 of SEQ ID NO: 64;

amino acids 90 to 88 of SEQ ID NO: 64;

amino acids 24 to 34 of SEQ ID NO: 66;

amino acids 50 to 57 of SEQ ID NO: 66;

amino acids 89 to 97 of SEQ ID NO: 66;

amino acids 24 to 34 of SEQ ID NO: 68;

amino acids 50 to 56 of SEQ ID NO: 68; and

amino acids 89 to 97 of SEQ ID NO: 68;

wherein the polypeptide, in association with an antibody heavy chain,binds TR-2.

In certain embodiments, an isolated polynucleotide is providedcomprising a sequence encoding a polypeptide comprising at least onecomplementarity determining region (CDR) selected from CDR1a, CDR2a, andCDR3a:

-   -   wherein CDR1a comprises the amino acid sequence a b c d e f g h        i j k l, wherein amino acid a is glycine, amino acid b is        selected from glycine, tyrosine, or phenylalanine; amino acid c        is selected from serine or threonine; amino acid d is selected        from isoleucine or phenylalanine; amino acid e is selected from        serine, threonine, or asparagine; amino acid f is selected from        serine, aspartic acid, tyrosine, asparagine, threonine, or        glycine; amino acid g is selected from glycine, aspartic acid,        or tyrosine; amino acid h is selected from glycine, aspartic        acid, tyrosine, asparagine, or serine; amino acid i is selected        from tyrosine, isoleucine, histidine, methionine, or tryptophan;        amino acid j is selected from asparagine, tyrosine, histidine,        serine, or phenylalanine; amino acid k is tryptophan or is not        present; and amino acid l is serine or is not present;    -   wherein CDR2a comprises the amino acid sequence m n o p q r t u        v w x y z a′ b′ c′, wherein amino acid m is selected from        tryptophan, tyrosine, histidine, valine, glutamic acid, or        serine; amino acid n is selected from methionine or isoleucine;        amino acid o is selected from asparagine, tyrosine, serine,        tryptophan, or histidine; amino acid p is selected from proline,        tyrosine, serine, arginine, histidine, or asparagine; amino acid        q is selected from asparagine, serine, or aspartic acid; amino        acid r is selected from serine or glycine; amino acid s is        selected from aspartic acid, serine, threonine, or arginine;        amino acid t is selected from asparagine, threonine, alanine,        isoleucine, or tyrosine; amino acid u is selected from        threonine, tyrosine, leucine, lysine, asparagine, or isoleucine;        amino acid v is selected from glycine, tyrosine, aspartic acid,        or cysteine; amino acid w is selected from tyrosine or        asparagine; amino acid x is selected from alanine or proline;        amino acid y is selected from glutamine, serine, or aspartic        acid; amino acid z is selected from lysine, leucine, or serine;        amino acid a′ is selected from phenylalanine, lysine, or valine;        amino acid b′ is selected from glutamine, serine, or lysine; and        amino acid c′ is glycine or is not present;    -   wherein CDR3a comprises the amino acid sequence d′ e′ f′ g′ h′        i′ j′ k′ l′ m′ n′ o′ p′ q′ r′ s′ t′ u′ v′ w′, wherein amino acid        d′ is selected from tryptophan, aspartic acid, glycine, serine,        or glutamic acid; amino acid e′ is selected from asparagine,        aspartic acid, glycine, arginine, serine, valine, or leucine;        amino acid f′ is selected from histidine, serine, alanine,        tyrosine, proline, asparagine, glycine or threonine; amino acid        g′ is selected from tyrosine, serine, alanine, arginine,        tryptophan, glycine or valine; amino acid h′ is selected from        glycine, alanine, serine, asparagine, methionine, tyrosine,        tryptophan, cysteine, or aspartic acid; amino acid i′ is        selected from serine, tryptophan, glycine, phenylalanine,        aspartic acid, tyrosine, or threonine; amino acid j′ is selected        from glycine, threonine, serine, leucine, valine, asparagine,        tryptophan, or tyrosine; amino acid k′ is selected from serine,        phenylalanine, aspartic acid, tryptophan, glycine, or tyrosine,        or is not present; amino acid l′ is selected from histidine,        aspartic acid, alanine, tryptophan, tyrosine, serine,        phenylalanine, valine, or glycine, or is not present; amino acid        m′ is selected from phenylalanine, tyrosine, glutamic acid,        proline, aspartic acid, cysteine, isoleucine, or methionine, or        is not present; amino acid n′ is selected from aspartic acid,        phenylalanine, alanine, leucine, or serine, or is not present;        amino acid o′ is selected from tyrosine, leucine, aspartic acid,        phenylalanine, proline, or valine, or is not present; amino acid        p′ is selected from leucine, aspartic acid, or tyrosine, or is        not present; amino acid q′ is selected from serine or tyrosine,        or is not present; amino acid r′ is tyrosine or is not present;        amino acid s′ is selected from glycine or tyrosine, or is not        present; amino acid t′ is selected from glycine or methionine,        or is not present; amino acid u′ is selected from methionine or        aspartic acid, or is not present; amino acid v′ is selected from        aspartic acid or valine, or is not present; and amino acid w′ is        valine or is not present; and        wherein the polypeptide, in association with an antibody light        chain, binds TR-2.

In certain embodiments, an isolated polynucleotide is providedcomprising a sequence encoding a polypeptide comprising at least onecomplementarity determining region (CDR) selected from CDR1b, CDR2b, andCDR3b:

-   -   wherein CDR1b comprises the amino acid sequence a1 b1 c1 d1 e1        f1 g1 h1 i1 j1 k1 l1 m1 n1 o1 p1 q1, wherein amino acid a1 is        selected from arginine or lysine; amino acid b1 is selected from        threonine, alanine, or serine; amino acid c1 is serine; amino        acid d1 is glutamine; amino acid e1 is selected from serine or        glycine; amino acid f1 is selected from isoleucine, leucine, or        valine; amino acid g1 is selected from serine, leucine, or        arginine; amino acid h1 is selected from threonine, serine,        isoleucine, asparagine, arginine, histidine, or tyrosine; amino        acid i1 is selected from tyrosine, arginine, tryptophan,        aspartic acid, or serine; j1 is selected from leucine,        isoleucine, asparagine, tyrosine, or serine; amino acid k1 is        selected from asparagine, glycine, valine, alanine, or leucine;        amino acid l1 is selected from tyrosine, alanine, or asparagine,        or is not present; amino acid m1 is selected from asparagine or        lysine, or is not present; amino acid n1 is selected from        tyrosine, asparagine, or isoleucine, or is not present; amino        acid o1 is selected from leucine or tyrosine, or is not present;        amino acid p1 is selected from aspartic acid or leucine, or is        not present; and amino acid q1 is selected from valine, alanine,        or threonine, or is not present;    -   wherein CDR2b comprises the amino acid sequence r1 s1 t1 u1 v1        w1 x1, wherein amino acid r1 is selected from alanine, aspartic        acid, leucine, tryptophan, glycine, or valine; amino acid s1 is        selected from threonine, valine, glycine, or alanine; amino acid        t1 is serine; amino acid u1 is selected from serine, asparagine,        or threonine; amino acid v1 is selected from leucine,        phenylalanine, or arginine; amino acid w1 is selected from        glutamine, alanine, or glutamic acid; and amino acid x1 is        selected from serine, arginine, or threonine; wherein CDR3b        comprises the amino acid sequence y1 z1 a1′ b1′ c1′ d1′ e1′ f1′        g1′, wherein amino acid y1 is selected from glutamine,        methionine, leucine, or histidine; amino acid z1 is selected        from glutamine or lysine; amino acid a1′ is selected from        serine, threonine, alanine, histidine, tyrosine, or        phenylalanine; amino acid b1′ is selected from tyrosine,        leucine, asparagine, or glycine; amino acid c1′ is selected from        serine, glutamine, isoleucine, or lysine; amino acid d1′ is        selected from threonine, phenylalanine, tyrosine, alanine, or        serine; amino acid e1′ is proline; amino acid f1′ is selected        from leucine, phenylalanine, tryptophan, serine, or arginine;        and amino acid g1′ is selected from threonine or serine; and    -   wherein the polypeptide, in association with an antibody heavy        chain, binds TR-2.

In certain embodiments, an isolated anti-TR-2 antibody comprising avariable region and a constant region is provided, wherein the antibodycomprises:

-   -   (i) a first polypeptide comprising at least one complementarity        determining region (CDR) selected from CDR1a, CDR2a, and CDR3a,        -   wherein CDR1a comprises the amino acid sequence a b c d e f            g h i j k l, wherein amino acid a is glycine, amino acid b            is selected from glycine, tyrosine, or phenylalanine; amino            acid c is selected from serine or threonine; amino acid d is            selected from isoleucine or phenylalanine; amino acid e is            selected from serine, threonine, or asparagine; amino acid f            is selected from serine, aspartic acid, tyrosine,            asparagine, threonine, or glycine; amino acid g is selected            from glycine, aspartic acid, or tyrosine; amino acid h is            selected from glycine, aspartic acid, tyrosine, asparagine,            or serine; amino acid i is selected from tyrosine,            isoleucine, histidine, methionine, or tryptophan; amino acid            j is selected from asparagine, tyrosine, histidine, serine,            or phenylalanine; amino acid k is tryptophan or is not            present; and amino acid l is serine or is not present;        -   wherein CDR2a comprises the amino acid sequence m n o p q r            t u v w x y z a′ b′ c′, wherein amino acid m is selected            from tryptophan, tyrosine, histidine, valine, glutamic acid,            or serine; amino acid n is selected from methionine or            isoleucine; amino acid o is selected from asparagine,            tyrosine, serine, tryptophan, or histidine; amino acid p is            selected from proline, tyrosine, serine, arginine,            histidine, or asparagine; amino acid q is selected from            asparagine, serine, or aspartic acid; amino acid r is            selected from serine or glycine; amino acid s is selected            from aspartic acid, serine, threonine, or arginine; amino            acid t is selected from asparagine, threonine, alanine,            isoleucine, or tyrosine; amino acid u is selected from            threonine, tyrosine, leucine, lysine, asparagine, or            isoleucine; amino acid v is selected from glycine, tyrosine,            aspartic acid, or cysteine; amino acid w is selected from            tyrosine or asparagine; amino acid x is selected from            alanine or proline; amino acid y is selected from glutamine,            serine, or aspartic acid; amino acid z is selected from            lysine, leucine, or serine; amino acid a′ is selected from            phenylalanine, lysine, or valine; amino acid b′ is selected            from glutamine, serine, or lysine; and amino acid c′ is            glycine or is not present;        -   wherein CDR3a comprises the amino acid sequence d′ e′ f′ g′            h′ i′ j′ k′ l′ m′ n′ o′ p′ q′ r′ s′ t′ u′ v′ w′, wherein            amino acid d′ is selected from tryptophan, aspartic acid,            glycine, serine, or glutamic acid; amino acid e′ is selected            from asparagine, aspartic acid, glycine, arginine, serine,            valine, or leucine; amino acid f′ is selected from            histidine, serine, alanine, tyrosine, proline, asparagine,            glycine or threonine; amino acid g′ is selected from            tyrosine, serine, alanine, arginine, tryptophan, glycine or            valine; amino acid h′ is selected from glycine, alanine,            serine, asparagine, methionine, tyrosine, tryptophan,            cysteine, or aspartic acid; amino acid i′ is selected from            serine, tryptophan, glycine, phenylalanine, aspartic acid,            tyrosine, or threonine; amino acid j′ is selected from            glycine, threonine, serine, leucine, valine, asparagine,            tryptophan, or tyrosine; amino acid k′ is selected from            serine, phenylalanine, aspartic acid, tryptophan, glycine,            or tyrosine, or is not present; amino acid l′ is selected            from histidine, aspartic acid, alanine, tryptophan,            tyrosine, serine, phenylalanine, valine, or glycine, or is            not present; amino acid m′ is selected from phenylalanine,            tyrosine, glutamic acid, proline, aspartic acid, cysteine,            isoleucine, or methionine, or is not present; amino acid n′            is selected from aspartic acid, phenylalanine, alanine,            leucine, or serine, or is not present; amino acid o′ is            selected from tyrosine, leucine, aspartic acid,            phenylalanine, proline, or valine, or is not present; amino            acid p′ is selected from leucine, aspartic acid, or            tyrosine, or is not present; amino acid q′ is selected from            serine or tyrosine, or is not present; amino acid r′ is            tyrosine or is not present; amino acid s′ is selected from            glycine or tyrosine, or is not present; amino acid t′ is            selected from glycine or methionine, or is not present;            amino acid u′ is selected from methionine or aspartic acid,            or is not present; amino acid v′ is selected from aspartic            acid or valine, or is not present; and amino acid w′ is            valine or is not present; and        -   wherein the first polypeptide, in association with an            antibody light chain, binds TR-2; and    -   (ii) a second polypeptide comprising at least one        complementarity determining region (CDR) selected from CDR1b,        CDR2b, and CDR3b        -   wherein CDR1b comprises the amino acid sequence a1 b1 c1 d1            e1 f1 g1 h1 i1 j1 k1 l1 m1 n1 o1 p1 q1, wherein amino acid            a1 is selected from arginine or lysine; amino acid b1 is            selected from threonine, alanine, or serine; amino acid c1            is serine; amino acid d1 is glutamine; amino acid e1 is            selected from serine or glycine; amino acid f1 is selected            from isoleucine, leucine, or valine; amino acid g1 is            selected from serine, leucine, or arginine; amino acid h1 is            selected from threonine, serine, isoleucine, asparagine,            arginine, histidine, or tyrosine; amino acid i1 is selected            from tyrosine, arginine, tryptophan, aspartic acid, or            serine; j1 is selected from leucine, isoleucine, asparagine,            tyrosine, or serine; amino acid k1 is selected from            asparagine, glycine, valine, alanine, or leucine; amino acid            l1 is selected from tyrosine, alanine, or asparagine, or is            not present; amino acid m1 is selected from asparagine or            lysine, or is not present; amino acid n1 is selected from            tyrosine, asparagine, or isoleucine, or is not present;            amino acid o1 is selected from leucine or tyrosine, or is            not present; amino acid p1 is selected from aspartic acid or            leucine, or is not present; and amino acid q1 is selected            from valine, alanine, or threonine, or is not present;        -   wherein CDR2b comprises the amino acid sequence r1 s1 t1 u1            v1 w1 x1, wherein amino acid r1 is selected from alanine,            aspartic acid, leucine, tryptophan, glycine, or valine;            amino acid s1 is selected from threonine, valine, glycine,            or alanine; amino acid t1 is serine; amino acid u1 is            selected from serine, asparagine, or threonine; amino acid            v1 is selected from leucine, phenylalanine, or arginine;            amino acid w1 is selected from glutamine, alanine, or            glutamic acid; and amino acid x1 is selected from serine,            arginine, or threonine;        -   wherein CDR3b comprises the amino acid sequence y1 z1 a1′            b1′ c1′ d1′ e1′ f1′ g1′, wherein amino acid y1 is selected            from glutamine, methionine, leucine, or histidine; amino            acid z1 is selected from glutamine or lysine; amino acid a1′            is selected from serine, threonine, alanine, histidine,            tyrosine, or phenylalanine; amino acid b1′ is selected from            tyrosine, leucine, asparagine, or glycine; amino acid c1′ is            selected from serine, glutamine, isoleucine, or lysine;            amino acid d1′ is selected from threonine, phenylalanine,            tyrosine, alanine, or serine; amino acid e1′ is proline;            amino acid f1′ is selected from leucine, phenylalanine,            tryptophan, serine, or arginine; and amino acid g1′ is            selected from threonine or serine; and        -   wherein the second polypeptide, in association with an            antibody heavy chain, binds TR-2.

In certain embodiments, an isolated anti-TR-2 antibody comprising avariable region and a constant region is provided, wherein the antibodycomprises:

-   -   a first polypeptide comprising complementarity determining        regions (CDRs) as set forth in SEQ ID NO: 2 and a second        polypeptide comprising CDRs as set forth in SEQ ID NO: 36; a        first polypeptide comprising complementarity determining regions        (CDRs) as set forth in SEQ ID NO: 4 and a second polypeptide        comprising CDRs as set forth in SEQ ID NO: 38; a first        polypeptide comprising complementarity determining regions        (CDRs) as set forth in SEQ ID NO: 6 and a second polypeptide        comprising CDRs as set forth in SEQ ID NO: 40; a first        polypeptide comprising complementarity determining regions        (CDRs) as set forth in SEQ ID NO: 8 and a second polypeptide        comprising CDRs as set forth in SEQ ID NO: 42; a first        polypeptide comprising complementarity determining regions        (CDRs) as set forth in SEQ ID NO: 10 and a second polypeptide        comprising CDRs as set forth in SEQ ID NO: 44; a first        polypeptide comprising complementarity determining regions        (CDRs) as set forth in SEQ ID NO: 12 and a second polypeptide        comprising CDRs as set forth in SEQ ID NO: 46; a first        polypeptide comprising complementarity determining regions        (CDRs) as set forth in SEQ ID NO: 14 and a second polypeptide        comprising CDRs as set forth in SEQ ID NO: 48; a first        polypeptide comprising complementarity determining regions        (CDRs) as set forth in SEQ ID NO: 16 and a second polypeptide        comprising CDRs as set forth in SEQ ID NO: 50; a first        polypeptide comprising complementarity determining regions        (CDRs) as set forth in SEQ ID NO: 18 and a second polypeptide        comprising CDRs as set forth in SEQ ID NO: 52; a first        polypeptide comprising complementarity determining regions        (CDRs) as set forth in SEQ ID NO: 20 and a second polypeptide        comprising CDRs as set forth in SEQ ID NO: 54; a first        polypeptide comprising complementarity determining regions        (CDRs) as set forth in SEQ ID NO: 22 and a second polypeptide        comprising CDRs as set forth in SEQ ID NO: 56; a first        polypeptide comprising complementarity determining regions        (CDRs) as set forth in SEQ ID NO: 24 and a second polypeptide        comprising CDRs as set forth in SEQ ID NO: 58; a first        polypeptide comprising complementarity determining regions        (CDRs) as set forth in SEQ ID NO: 26 and a second polypeptide        comprising CDRs as set forth in SEQ ID NO: 60; a first        polypeptide comprising complementarity determining regions        (CDRs) as set forth in SEQ ID NO: 28 and a second polypeptide        comprising CDRs as set forth in SEQ ID NO: 62; a first        polypeptide comprising complementarity determining regions        (CDRs) as set forth in SEQ ID NO: 30 and a second polypeptide        comprising CDRs as set forth in SEQ ID NO: 64; a first        polypeptide comprising complementarity determining regions        (CDRs) as set forth in SEQ ID NO: 32 and a second polypeptide        comprising CDRs as set forth in SEQ ID NO: 66; or a first        polypeptide comprising complementarity determining regions        (CDRs) as set forth in SEQ ID NO: 34 and a second polypeptide        comprising CDRs as set forth in SEQ ID NO: 68.

In certain embodiments, a cell is provided, comprising:

-   -   (a) a first polynucleotide comprising a sequence encoding a        first polypeptide comprising at least one complementarity        determining region (CDR) selected from CDR1a, CDR2a, and CDR3a,        wherein CDR1a comprises the amino acid sequence a b c d e f g h        i j k l, wherein amino acid a is glycine, amino acid b is        selected from glycine, tyrosine, or phenylalanine; amino acid c        is selected from serine or threonine; amino acid d is selected        from isoleucine or phenylalanine; amino acid e is selected from        serine, threonine, or asparagine; amino acid f is selected from        serine, aspartic acid, tyrosine, asparagine, threonine, or        glycine; amino acid g is selected from glycine, aspartic acid,        or tyrosine; amino acid h is selected from glycine, aspartic        acid, tyrosine, asparagine, or serine; amino acid i is selected        from tyrosine, isoleucine, histidine, methionine, or tryptophan;        amino acid j is selected from asparagine, tyrosine, histidine,        serine, or phenylalanine; amino acid k is tryptophan or is not        present; and amino acid l is serine or is not present;    -   wherein CDR2a comprises the amino acid sequence m n o p q r t u        v w x y z a′ b′ c′, wherein amino acid m is selected from        tryptophan, tyrosine, histidine, valine, glutamic acid, or        serine; amino acid n is selected from methionine or isoleucine;        amino acid o is selected from asparagine, tyrosine, serine,        tryptophan, or histidine; amino acid p is selected from proline,        tyrosine, serine, arginine, histidine, or asparagine; amino acid        q is selected from asparagine, serine, or aspartic acid; amino        acid r is selected from serine or glycine; amino acid s is        selected from aspartic acid, serine, threonine, or arginine;        amino acid t is selected from asparagine, threonine, alanine,        isoleucine, or tyrosine; amino acid u is selected from        threonine, tyrosine, leucine, lysine, asparagine, or isoleucine;        amino acid v is selected from glycine, tyrosine, aspartic acid,        or cysteine; amino acid w is selected from tyrosine or        asparagine; amino acid x is selected from alanine or proline;        amino acid y is selected from glutamine, serine, or aspartic        acid; amino acid z is selected from lysine, leucine, or serine;        amino acid a′ is selected from phenylalanine, lysine, or valine;        amino acid b′ is selected from glutamine, serine, or lysine; and        amino acid c′ is glycine or is not present;    -   wherein CDR3a comprises the amino acid sequence d′ e′ f′ g′ h′        i′ j′ k′ l′ m′ n′ o′ p′ q′ r′ s′ t′ u′ v′ w′, wherein amino acid        d′ is selected from tryptophan, aspartic acid, glycine, serine,        or glutamic acid; amino acid e′ is selected from asparagine,        aspartic acid, glycine, arginine, serine, valine, or leucine;        amino acid f′ is selected from histidine, serine, alanine,        tyrosine, proline, asparagine, glycine or threonine; amino acid        g′ is selected from tyrosine, serine, alanine, arginine,        tryptophan, glycine or valine; amino acid h′ is selected from        glycine, alanine, serine, asparagine, methionine, tyrosine,        tryptophan, cysteine, or aspartic acid; amino acid i′ is        selected from serine, tryptophan, glycine, phenylalanine,        aspartic acid, tyrosine, or threonine; amino acid j′ is selected        from glycine, threonine, serine, leucine, valine, asparagine,        tryptophan, or tyrosine; amino acid k′ is selected from serine,        phenylalanine, aspartic acid, tryptophan, glycine, or tyrosine,        or is not present; amino acid l′ is selected from histidine,        aspartic acid, alanine, tryptophan, tyrosine, serine,        phenylalanine, valine, or glycine, or is not present; amino acid        m′ is selected from phenylalanine, tyrosine, glutamic acid,        proline, aspartic acid, cysteine, isoleucine, or methionine, or        is not present; amino acid n′ is selected from aspartic acid,        phenylalanine, alanine, leucine, or serine, or is not present;        amino acid o′ is selected from tyrosine, leucine, aspartic acid,        phenylalanine, proline, or valine, or is not present; amino acid        p′ is selected from leucine, aspartic acid, or tyrosine, or is        not present; amino acid q′ is selected from serine or tyrosine,        or is not present; amino acid r′ is tyrosine or is not present;        amino acid s′ is selected from glycine or tyrosine, or is not        present; amino acid t′ is selected from glycine or methionine,        or is not present; amino acid u′ is selected from methionine or        aspartic acid, or is not present; amino acid v′ is selected from        aspartic acid or valine, or is not present; and amino acid w′ is        valine or is not present; wherein the first polypeptide, in        association with an antibody light chain, binds TR-2; and    -   (b) a second polynucleotide comprising a sequence encoding a        second polypeptide comprising at least one complementarity        determining region (CDR) selected from CDR1b, CDR2b, and CDR3b,        -   wherein CDR1b comprises the amino acid sequence a1 b1 c1 d1            e1 f1 g1 h1 i1 j1 k1 l1 m1 n1 o1 p1 q1, wherein amino acid            a1 is selected from arginine or lysine; amino acid b1 is            selected from threonine, alanine, or serine; amino acid c1            is serine; amino acid d1 is glutamine; amino acid e1 is            selected from serine or glycine; amino acid f1 is selected            from isoleucine, leucine, or valine; amino acid g1 is            selected from serine, leucine, or arginine; amino acid h1 is            selected from threonine, serine, isoleucine, asparagine,            arginine, histidine, or tyrosine; amino acid i1 is selected            from tyrosine, arginine, tryptophan, aspartic acid, or            serine; j1 is selected from leucine, isoleucine, asparagine,            tyrosine, or serine; amino acid k1 is selected from            asparagine, glycine, valine, alanine, or leucine; amino acid            l1 is selected from tyrosine, alanine, or asparagine, or is            not present; amino acid m1 is selected from asparagine or            lysine, or is not present; amino acid n1 is selected from            tyrosine, asparagine, or isoleucine, or is not present;            amino acid o1 is selected from leucine or tyrosine, or is            not present; amino acid p1 is selected from aspartic acid or            leucine, or is not present; and amino acid q1 is selected            from valine, alanine, or threonine, or is not present;        -   wherein CDR2b comprises the amino acid sequence r1 s1 t1 u1            v1 w1 x1, wherein amino acid r1 is selected from alanine,            aspartic acid, leucine, tryptophan, glycine, or valine;            amino acid s1 is selected from threonine, valine, glycine,            or alanine; amino acid t1 is serine; amino acid u1 is            selected from serine, asparagine, or threonine; amino acid            v1 is selected from leucine, phenylalanine, or arginine;            amino acid w1 is selected from glutamine, alanine, or            glutamic acid; and amino acid x1 is selected from serine,            arginine, or threonine;    -   wherein CDR3b comprises the amino acid sequence y1 z1 a1′ b1′        c1′ d1′ e1′ f1′ g1′, wherein amino acid y1 is selected from        glutamine, methionine, leucine, or histidine; amino acid z1 is        selected from glutamine or lysine; amino acid a1′ is selected        from serine, threonine, alanine, histidine, tyrosine, or        phenylalanine; amino acid b1′ is selected from tyrosine,        leucine, asparagine, or glycine; amino acid c1′ is selected from        serine, glutamine, isoleucine, or lysine; amino acid d1′ is        selected from threonine, phenylalanine, tyrosine, alanine, or        serine; amino acid e1′ is proline; amino acid f1′ is selected        from leucine, phenylalanine, tryptophan, serine, or arginine;        and amino acid g1′ is selected from threonine or serine; wherein        the second polypeptide, in association with an antibody heavy        chain, binds TR-2.

In certain embodiments, an isolated antibody is provided thatspecifically binds to an epitope that is specifically bound by at leastone antibody selected from: Ab A, Ab B, Ab C, Ab D, Ab E, Ab F, Ab G, AbH, Ab I, Ab J, Ab K, Ab L, Ab M, Ab N, Ab O, Ab P, and Ab Q.

In certain embodiments, a polypeptide is provided comprising at leastone amino acid sequence selected from SEQ ID NO: 94, SEQ ID NO: 95, andSEQ ID NO: 96.

In certain embodiments, a polypeptide is provided consisting essentiallyof at least one amino acid sequence selected from SEQ ID NO: 94, SEQ IDNO: 95, and SEQ ID NO: 96.

In certain embodiments, an antibody or antigen binding domain isprovided which binds at least one amino acid sequence selected from SEQID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96.

In certain embodiments, a method of obtaining an antibody that bindsTR-2 is provided comprising administering at least one polypeptideselected from SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96 to ananimal and obtaining an antibody that binds TR-2 from the animal.

In certain embodiments, a method of decreasing or preventing binding ofan antibody to TR-2 by administering a polypeptide comprising at leastone amino acid sequence selected from SEQ ID NO: 94, SEQ ID NO: 95, andSEQ ID NO: 96 is provided.

In certain embodiments, a method of decreasing or preventing binding ofan antibody to TR-2 by administering a polypeptide consisting of atleast one amino acid sequence selected from SEQ ID NO: 94, SEQ ID NO:95, and SEQ ID NO: 96 is provided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the immunization schedule used in Example 1 for a TR-2-Hisconstruct in transgenic mice expressing human immunoglobulin genes, viaeither footpad inoculation (groups 1, 2, and 3) or via intraperitonealinjection (groups 4 and 5).

FIG. 2 shows the results of an ELISA assay to measure the reactivity ofcertain blood samples from selected mice described in FIG. 1 to theantigen TR-2, according to work described in Example 1.

FIG. 3 shows the nucleotide sequences encoding the heavy chain (SEQ IDNO: 1) and light chain (SEQ ID NO: 35) variable regions of anti-TR-2antibody A, and the amino acid sequences of the heavy chain (SEQ ID NO:2) and the light chain (SEQ ID NO: 36) variable regions of thatantibody.

FIG. 4 shows the nucleotide sequences encoding the heavy chain (SEQ IDNO: 3) and light chain (SEQ ID NO: 37) variable regions of anti-TR-2antibody B, and the amino acid sequences of the heavy chain (SEQ ID NO:4) and the light chain (SEQ ID NO: 38) variable regions of thatantibody.

FIG. 5 shows the nucleotide sequences encoding the heavy chain (SEQ IDNO: 5) and light chain (SEQ ID NO: 39) variable regions of anti-TR-2antibody C, and the amino acid sequences of the heavy chain (SEQ ID NO:6) and the light chain (SEQ ID NO: 40) variable regions of thatantibody.

FIG. 6 shows the nucleotide sequences encoding the heavy chain (SEQ IDNO: 7) and light chain (SEQ ID NO: 41) variable regions of anti-TR-2antibody D, and the amino acid sequences of the heavy chain (SEQ ID NO:8) and the light chain (SEQ ID NO: 42) variable regions of thatantibody.

FIG. 7 shows the nucleotide sequences encoding the heavy chain (SEQ IDNO: 9) and light chain (SEQ ID NO: 43) variable regions of anti-TR-2antibody E, and the amino acid sequences of the heavy chain (SEQ ID NO:10) and the light chain (SEQ ID NO: 44) variable regions of thatantibody.

FIG. 8 shows the nucleotide sequences encoding the heavy chain (SEQ IDNO: 11) and light chain (SEQ ID NO: 45) variable regions of anti-TR-2antibody F, and the amino acid sequences of the heavy chain (SEQ ID NO:12) and the light chain (SEQ ID NO: 46) variable regions of thatantibody.

FIG. 9 shows the nucleotide sequences encoding the heavy chain (SEQ IDNO: 13) and light chain (SEQ ID NO: 47) variable regions of anti-TR-2antibody G, and the amino acid sequences of the heavy chain (SEQ ID NO:14) and the light chain (SEQ ID NO: 48) variable regions of thatantibody.

FIG. 10 shows the nucleotide sequences encoding the heavy chain (SEQ IDNO: 15) and light chain (SEQ ID NO: 49) variable regions of anti-TR-2antibody H, and the amino acid sequences of the heavy chain (SEQ ID NO:16) and the light chain (SEQ ID NO: 50) variable regions of thatantibody.

FIG. 11 shows the nucleotide sequences encoding the heavy chain (SEQ IDNO: 17) and light chain (SEQ ID NO: 51) variable regions of anti-TR-2antibody 1, and the amino acid sequences of the heavy chain (SEQ ID NO:18) and the light chain (SEQ ID NO: 52) variable regions of thatantibody.

FIG. 12 shows the nucleotide sequences encoding the heavy chain (SEQ IDNO: 19) and light chain (SEQ ID NO: 53) variable regions of anti-TR-2antibody J, and the amino acid sequences of the heavy chain (SEQ ID NO:20) and the light chain (SEQ ID NO: 54) variable regions of thatantibody.

FIG. 13 shows the nucleotide sequences encoding the heavy chain (SEQ IDNO: 21) and light chain (SEQ ID NO: 55) variable regions of anti-TR-2antibody K, and the amino acid sequences of the heavy chain (SEQ ID NO:22) and the light chain (SEQ ID NO: 56) variable regions of thatantibody.

FIG. 14 shows the nucleotide sequences encoding the heavy chain (SEQ IDNO: 23) and light chain (SEQ ID NO: 57) variable regions of anti-TR-2antibody L, and the amino acid sequences of the heavy chain (SEQ ID NO:24) and the light chain (SEQ ID NO: 58) variable regions of thatantibody.

FIG. 15 shows the nucleotide sequences encoding the heavy chain (SEQ IDNO: 25) and light chain (SEQ ID NO: 59) variable regions of anti-TR-2antibody M, and the amino acid sequences of the heavy chain (SEQ ID NO:26) and the light chain (SEQ ID NO: 60) variable regions of thatantibody.

FIG. 16 shows the nucleotide sequences encoding the heavy chain (SEQ IDNO: 27) and light chain (SEQ ID NO: 61) variable regions of anti-TR-2antibody N, and the amino acid sequences of the heavy chain (SEQ ID NO:28) and the light chain (SEQ ID NO: 62) variable regions of thatantibody.

FIG. 17 shows the nucleotide sequences encoding the heavy chain (SEQ IDNO: 29) and light chain (SEQ ID NO: 63) variable regions of anti-TR-2antibody 0, and the amino acid sequences of the heavy chain (SEQ ID NO:30) and the light chain (SEQ ID NO: 64) variable regions of thatantibody.

FIG. 18 shows the nucleotide sequences encoding the heavy chain (SEQ IDNO: 31) and light chain (SEQ ID NO: 65) variable regions of anti-TR-2antibody P, and the amino acid sequences of the heavy chain (SEQ ID NO:32) and the light chain (SEQ ID NO: 66) variable regions of thatantibody.

FIG. 19 shows the nucleotide sequences encoding the heavy chain (SEQ IDNO: 33) and light chain (SEQ ID NO: 67) variable regions of anti-TR-2antibody Q, and the amino acid sequences of the heavy chain (SEQ ID NO:34) and the light chain (SEQ ID NO: 68) variable regions of thatantibody.

FIG. 20 is an alignment of the amino acid sequences of the heavy chainvariable regions for anti-TR-2 antibodies A to Q (SEQ ID NOs: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34). Frameworkregions 1 through 3 (FR1, FR2, and FR3) and complementarity determiningregions 1 through 3 (CDR1, CDR2, and CDR3) for each sequence are shown.

FIG. 21 is an alignment of the amino acid sequences of the light chainvariable regions for anti-TR-2 antibodies A to Q (SEQ ID NOs: 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, and 68).Framework regions 1 through 3 (FR1, FR2, and FR3) and complementaritydetermining regions 1 through 3 (CDR1, CDR2, and CDR3) for each sequenceare shown.

FIG. 22 is a table showing the classification of certain human anti-TR-2antibodies into one of four reactivity groups according to the abilityof each to bind to the truncated and chimeric N-avidin TR-2 proteins,according to work described in Example 5.

FIG. 23 shows schematic representations of the thirteen truncations ofhuman N-avidin-TR-2 used in epitope mapping, according to the workdescribed in Example 6.

FIG. 24 is a bar graph showing the binding of certain human anti-TR-2antibodies to the N-avidin-TR-2 truncations according to work describedin Example 6.

FIG. 25 shows schematic representations of N-avidin-cyno TR-2truncations and N-avidin-cyno/human TR-2 chimeras used in epitopemapping, according to work described in Example 6.

FIG. 26 is an alignment of the human TR-2, cyno TR-2 (short form), andmouse TR-2 sequences, according to work described in Example 6.

FIG. 27 is a bar graph showing the binding of certain human anti-TR-2antibodies to the N-avidin-TR-2 truncations, chimeras, and domainreplacements according to work described in Example 6.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents or portions of documents cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are expressly incorporated by reference herein intheir entirety for any purpose.

DEFINITIONS

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See e.g., Sambrook et al. Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989)). Unless specific definitions are provided,the nomenclatures utilized in connection with, and the laboratoryprocedures and techniques of, analytical chemistry, synthetic organicchemistry, and medicinal and pharmaceutical chemistry described hereinare those well known and commonly used in the art. Standard techniquesmay be used for chemical syntheses, chemical analyses, pharmaceuticalpreparation, formulation, delivery, and treatment of patients.

In this application, the use of the singular includes the plural unlessspecifically stated otherwise. In this application, the use of “or”means “and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Also, terms such as “element” or “component” encompassboth elements and components comprising one unit and elements andcomponents that comprise more than one subunit unless specificallystated otherwise.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is linked to apolynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence.

The terms “polynucleotide” and “oligonucleotide” are usedinterchangeably, and as referred to herein mean a polymeric form ofnucleotides of at least 10 bases in length. In certain embodiments, thebases may comprise at least one of ribonucleotides,deoxyribonucleotides, and a modified form of either type of nucleotide.The term includes single and double stranded forms of DNA. The term“polynucleotide” also encompasses sequences that comprise one or more ofSEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, and67. In certain embodiments, polynucleotides have nucleotide sequencesthat are about 90 percent, or about 95 percent, or about 96 percent, orabout 97 percent, or about 98 percent, or about 99 percent identical tonucleotide sequences shown in FIGS. 3-19. In certain embodiments,polynucleotides complementary to specific polynucleotides that encodecertain polypeptides described herein are provided.

In certain embodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising at least one complementarity determining region(CDR) selected from CDR1a, CDR2a, and CDR3a, wherein CDR1a comprises theamino acid sequence a b c d e f g h i j k l, wherein amino acid a isglycine, amino acid b is selected from glycine, tyrosine, orphenylalanine; amino acid c is selected from serine or threonine; aminoacid d is selected from isoleucine or phenylalanine; amino acid e isselected from serine, threonine, or asparagine; amino acid f is selectedfrom serine, aspartic acid, tyrosine, asparagine, threonine, or glycine;amino acid g is selected from glycine, aspartic acid, or tyrosine; aminoacid h is selected from glycine, aspartic acid, tyrosine, asparagine, orserine; amino acid i is selected from tyrosine, isoleucine, histidine,methionine, or tryptophan; amino acid j is selected from asparagine,tyrosine, histidine, serine, or phenylalanine; amino acid k istryptophan or is not present; and amino acid l is serine or is notpresent; wherein CDR2a comprises the amino acid sequence m n o p q r t uv w x y z a′ b′ c′, wherein amino acid m is selected from tryptophan,tyrosine, histidine, valine, glutamic acid, or serine; amino acid n isselected from methionine or isoleucine; amino acid o is selected fromasparagine, tyrosine, serine, tryptophan, or histidine; amino acid p isselected from proline, tyrosine, serine, arginine, histidine, orasparagine; amino acid q is selected from asparagine, serine, oraspartic acid; amino acid r is selected from serine or glycine; aminoacid s is selected from aspartic acid, serine, threonine, or arginine;amino acid t is selected from asparagine, threonine, alanine,isoleucine, or tyrosine; amino acid u is selected from threonine,tyrosine, leucine, lysine, asparagine, or isoleucine; amino acid v isselected from glycine, tyrosine, aspartic acid, or cysteine; amino acidw is selected from tyrosine or asparagine; amino acid x is selected fromalanine or proline; amino acid y is selected from glutamine, serine, oraspartic acid; amino acid z is selected from lysine, leucine, or serine;amino acid a′ is selected from phenylalanine, lysine, or valine; aminoacid b′ is selected from glutamine, serine, or lysine; and amino acid c′is glycine or is not present; wherein CDR3a comprises the amino acidsequence d′ e′ f′ g′ h′ i′ j′ k′ l′ m′ n′ o′ p′ q′ r′ s′ t′ u′ v′ w′,wherein amino acid d′ is selected from tryptophan, aspartic acid,glycine, serine, or glutamic acid; amino acid e′ is selected fromasparagine, aspartic acid, glycine, arginine, serine, valine, orleucine; amino acid f′ is selected from histidine, serine, alanine,tyrosine, proline, asparagine, glycine or threonine; amino acid g′ isselected from tyrosine, serine, alanine, arginine, tryptophan, glycineor valine; amino acid h′ is selected from glycine, alanine, serine,asparagine, methionine, tyrosine, tryptophan, cysteine, or asparticacid; amino acid i′ is selected from serine, tryptophan, glycine,phenylalanine, aspartic acid, tyrosine, or threonine; amino acid j′ isselected from glycine, threonine, serine, leucine, valine, asparagine,tryptophan, or tyrosine; amino acid k′ is selected from serine,phenylalanine, aspartic acid, tryptophan, glycine, or tyrosine, or isnot present; amino acid l′ is selected from histidine, aspartic acid,alanine, tryptophan, tyrosine, serine, phenylalanine, valine, orglycine, or is not present; amino acid m′ is selected fromphenylalanine, tyrosine, glutamic acid, proline, aspartic acid,cysteine, isoleucine, or methionine, or is not present; amino acid n′ isselected from aspartic acid, phenylalanine, alanine, leucine, or serine,or is not present; amino acid o′ is selected from tyrosine, leucine,aspartic acid, phenylalanine, proline, or valine, or is not present;amino acid p′ is selected from leucine, aspartic acid, or tyrosine, oris not present; amino acid q′ is selected from serine or tyrosine, or isnot present; amino acid r′ is tyrosine or is not present; amino acid s′is selected from glycine or tyrosine, or is not present; amino acid t′is selected from glycine or methionine, or is not present; amino acid u′is selected from methionine or aspartic acid, or is not present; aminoacid v′ is selected from aspartic acid or valine, or is not present; andamino acid w′ is valine or is not present; and wherein the polypeptide,in association with an antibody light chain, binds TR-2.

In certain embodiments, a polynucleotide comprises a sequence encodingCDR2a, wherein CDR2a comprises the amino acid sequence m n o p q r s t uv w x y z a′ b′ c′, wherein amino acid m is selected from tryptophan,tyrosine, histidine, valine, glutamic acid, or serine; amino acid n isselected from methionine or isoleucine; amino acid o is selected fromasparagine, tyrosine, serine, tryptophan, or histidine; amino acid p isselected from proline, tyrosine, serine, arginine, histidine, orasparagine; amino acid q is selected from asparagine, serine, oraspartic acid; amino acid r is selected from serine or glycine; aminoacid s is selected from aspartic acid, serine, threonine, or arginine;amino acid t is selected from asparagine, threonine, alanine,isoleucine, or tyrosine; amino acid u is selected from threonine,tyrosine, leucine, lysine, asparagine, or isoleucine; amino acid v isselected from glycine, tyrosine, aspartic acid, or cysteine; amino acidw is selected from tyrosine or asparagine; amino acid x is selected fromalanine or proline; amino acid y is selected from glutamine, serine, oraspartic acid; amino acid z is selected from lysine, leucine, or serine;amino acid a′ is selected from phenylalanine, lysine, or valine; aminoacid b′ is selected from glutamine, serine, or lysine; and amino acid c′is glycine or is not present.

In certain embodiments, a polynucleotide comprises a sequence encodingCDR3a comprising the amino acid sequence d′ e′ f′ g′ h′ i′ j′ k′ l′ m′n′ o′ p′ q′ r′ s′ t′ u′ v′ w′, wherein amino acid d′ is selected fromtryptophan, aspartic acid, glycine, serine, or glutamic acid; amino acide′ is selected from asparagine, aspartic acid, glycine, arginine,serine, valine, or leucine; amino acid f′ is selected from histidine,serine, alanine, tyrosine, proline, asparagine, glycine or threonine;amino acid g′ is selected from tyrosine, serine, alanine, arginine,tryptophan, glycine or valine; amino acid h′ is selected from glycine,alanine, serine, asparagine, methionine, tyrosine, tryptophan, cysteine,or aspartic acid; amino acid i′ is selected from serine, tryptophan,glycine, phenylalanine, aspartic acid, tyrosine, or threonine; aminoacid j′ is selected from glycine, threonine, serine, leucine, valine,asparagine, tryptophan, or tyrosine; amino acid k′ is selected fromserine, phenylalanine, aspartic acid, tryptophan, glycine, or tyrosine,or is not present; amino acid l′ is selected from histidine, asparticacid, alanine, tryptophan, tyrosine, serine, phenylalanine, valine, orglycine, or is not present; amino acid m′ is selected fromphenylalanine, tyrosine, glutamic acid, proline, aspartic acid,cysteine, isoleucine, or methionine, or is not present; amino acid n′ isselected from aspartic acid, phenylalanine, alanine, leucine, or serine,or is not present; amino acid o′ is selected from tyrosine, leucine,aspartic acid, phenylalanine, proline, or valine, or is not present;amino acid p′ is selected from leucine, aspartic acid, or tyrosine, oris not present; amino acid q′ is selected from serine or tyrosine, or isnot present; amino acid r′ is tyrosine or is not present; amino acid s′is selected from glycine or tyrosine, or is not present; amino acid t′is selected from glycine or methionine, or is not present; amino acid u′is selected from methionine or aspartic acid, or is not present; aminoacid v′ is selected from aspartic acid or valine, or is not present; andamino acid w′ is valine or is not present.

In certain embodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising at least two complementarity determining regions(CDR) selected from CDR1a, CDR2a, and CDR3a, wherein the polypeptide, inassociation with an antibody light chain, binds TR-2. In certainembodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising CDR1a, CDR2a, and CDR3a, wherein the polypeptide,in association with an antibody light chain, binds TR-2.

In certain embodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising an antibody heavy chain variable region. Incertain embodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising a human antibody heavy chain variable region. Incertain embodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising a heavy chain constant region. In certainembodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising a human heavy chain constant region. In certainembodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ IDNO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQID NO: 32, or SEQ ID NO: 34. In certain embodiments, a polynucleotidecomprises a sequence encoding a polypeptide comprising a non-human heavychain constant region. In certain embodiments, a polynucleotidecomprises a sequence encoding a polypeptide comprising a heavy chainconstant region of a species other than human.

In certain embodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising at least one complementarity determining region(CDR) selected from amino acids 26 to 35 of SEQ ID NO: 2; amino acids 50to 66 of SEQ ID NO: 2; amino acids 99 to 110 of SEQ ID NO: 2; aminoacids 26 to 37 of SEQ ID NO: 4; amino acids 52 to 67 of SEQ ID NO: 4;amino acids 100 to 109 of SEQ ID NO: 4; amino acids 26 to 37 of SEQ IDNO: 6; amino acids 52 to 67 of SEQ ID NO: 6; amino acids 100 to 109 ofSEQ ID NO: 6; amino acids 26 to 37 of SEQ ID NO: 8; amino acids 52 to 67of SEQ ID NO: 8; amino acids 100 to 109 of SEQ ID NO: 8; amino acids 26to 35 of SEQ ID NO: 10; amino acids 50 to 66 of SEQ ID NO: 10; aminoacids 99 to 110 of SEQ ID NO: 10; amino acids 26 to 35 of SEQ ID NO: 12;amino acids 50 to 66 of SEQ ID NO: 12; amino acids 99 to 111 of SEQ IDNO: 12; amino acids 26 to 35 of SEQ ID NO: 14; amino acids 50 to 65 ofSEQ ID NO: 14; amino acids 98 to 111 of SEQ ID NO: 14; amino acids 26 to37 of SEQ ID NO: 16; amino acids 52 to 67 of SEQ ID NO: 16; amino acids100 to 109 of SEQ ID NO: 16; amino acids 26 to 35 of SEQ ID NO: 18;amino acids 50 to 66 of SEQ ID NO: 18; amino acids 99 to 105 of SEQ IDNO: 18; amino acids 26 to 35 of SEQ ID NO: 20; amino acids 50 to 66 ofSEQ ID NO: 20; amino acids 99 to 118 of SEQ ID NO: 20; amino acids 26 to35 of SEQ ID NO: 22; amino acids 50 to 66 of SEQ ID NO: 22; amino acids99 to 118 of SEQ ID NO: 22; amino acids 26 to 35 of SEQ ID NO: 24; aminoacids 50 to 65 of SEQ ID NO: 24; amino acids 98 to 108 of SEQ ID NO: 24;amino acids 26 to 35 of SEQ ID NO: 26; amino acids 50 to 66 of SEQ IDNO: 26; amino acids 99 to 110 of SEQ ID NO: 26; amino acids 26 to 35 ofSEQ ID NO: 28; amino acids 50 to 66 of SEQ ID NO: 28; amino acids 99 to117 of SEQ ID NO: 28; amino acids 26 to 37 of SEQ ID NO: 30; amino acids52 to 67 of SEQ ID NO: 30; amino acids 100 to 111 of SEQ ID NO: 30;amino acids 26 to 37 of SEQ ID NO: 32; amino acids 52 to 67 of SEQ IDNO: 32; amino acids 100 to 111 of SEQ ID NO: 32; amino acids 26 to 37 ofSEQ ID NO: 34; amino acids 52 to 67 of SEQ ID NO: 34; and amino acids100 to 111 of SEQ ID NO: 34, wherein the polypeptide, in associationwith an antibody light chain, binds TR-2. In certain embodiments, apolynucleotide comprises a sequence encoding a polypeptide comprising atleast two of the CDRs of SEQ ID NOS. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, or 34. In certain embodiments, a polynucleotidecomprises a sequence encoding a polypeptide comprising three of the CDRsof SEQ ID NOS. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, or 34.

In certain embodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising amino acids 26 to 35 of SEQ ID NO: 2, amino acids50 to 66 of SEQ ID NO: 2, and amino acids 99 to 110 of SEQ ID NO: 2. Incertain embodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising amino acids 26 to 37 of SEQ ID NO: 4, amino acids52 to 67 of SEQ ID NO: 4, and amino acids 100 to 109 of SEQ ID NO: 4. Incertain embodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising amino acids 26 to 37 of SEQ ID NO: 6, amino acids52 to 67 of SEQ ID NO: 6, and amino acids 100 to 109 of SEQ ID NO: 6. Incertain embodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising amino acids 26 to 37 of SEQ ID NO: 8, amino acids52 to 67 of SEQ ID NO: 8, and amino acids 100 to 109 of SEQ ID NO: 8. Incertain embodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising amino acids 26 to 35 of SEQ ID NO: 10, aminoacids 50 to 66 of SEQ ID NO: 10, and amino acids 99-110 of SEQ ID NO:10. In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising amino acids 26 to 35 of SEQ ID NO: 12,amino acids 50 to 66 of SEQ ID NO: 12, and amino acids 99-111 of SEQ IDNO: 12. In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising amino acids 26 to 35 of SEQ ID NO: 14,amino acids 50 to 65 of SEQ ID NO: 14, and amino acids 98 to 111 of SEQID NO: 14. In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising amino acids 26 to 37 of SEQ ID NO: 16,amino acids 52 to 67 of SEQ ID NO: 16, and amino acids 100 to 109 of SEQID NO: 16. In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising amino acids 26 to 35 of SEQ ID NO: 18,amino acids 50 to 66 of SEQ ID NO: 18, and amino acids 99 to 105 of SEQID NO: 18. In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising amino acids 26 to 35 of SEQ ID NO: 20,amino acids 50 to 66 of SEQ ID NO: 20, and amino acids 99 to 118 of SEQID NO: 20. In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising amino acids 26 to 35 of SEQ ID NO: 22,amino acids 50 to 66 of SEQ ID NO: 22, and amino acids 99 to 118 of SEQID NO: 22. In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising amino acids 26 to 35 of SEQ ID NO: 24,amino acids 50 to 65 of SEQ ID NO: 24, and amino acids 98 to 108 of SEQID NO: 24. In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising amino acids 26 to 35 of SEQ ID NO: 26,amino acids 50 to 66 of SEQ ID NO: 26, and amino acids 99 to 110 of SEQID NO: 26. In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising amino acids 26 to 35 of SEQ ID NO: 28,amino acids 50 to 66 of SEQ ID NO: 28, and amino acids 99 to 117 of SEQID NO: 28. In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising amino acids 26 to 37 of SEQ ID NO: 30,amino acids 52 to 67 of SEQ ID NO: 30, and amino acids 100 to 111 of SEQID NO: 30. In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising amino acids 26 to 37 of SEQ ID NO: 32,amino acids 52 to 67 of SEQ ID NO: 32, and amino acids 100 to 111 of SEQID NO: 32. In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising amino acids 26 to 37 of SEQ ID NO: 34,amino acids 52 to 67 of SEQ ID NO: 34, and amino acids 100 to 111 of SEQID NO: 34.

In certain embodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising at least one complementarity determining region(CDR) selected from CDR1b, CDR2b, and CDR3b, wherein CDR1b comprises theamino acid sequence a1 b1 c1 d1 e1 f1 g1 h1 i1 j1 k1 l1 m1 n1 o1 p1 q1,wherein amino acid a1 is selected from arginine or lysine; amino acid b1is selected from threonine, alanine, or serine; amino acid c1 is serine;amino acid d1 is glutamine; amino acid e1 is selected from serine orglycine; amino acid f1 is selected from isoleucine, leucine, or valine;amino acid g1 is selected from serine, leucine, or arginine; amino acidh1 is selected from threonine, serine, isoleucine, asparagine, arginine,histidine, or tyrosine; amino acid i1 is selected from tyrosine,arginine, tryptophan, aspartic acid, or serine; j1 is selected fromleucine, isoleucine, asparagine, tyrosine, or serine; amino acid k1 isselected from asparagine, glycine, valine, alanine, or leucine; aminoacid l1 is selected from tyrosine, alanine, or asparagine, or is notpresent; amino acid m1 is selected from asparagine or lysine, or is notpresent; amino acid n1 is selected from tyrosine, asparagine, orisoleucine, or is not present; amino acid o1 is selected from leucine ortyrosine, or is not present; amino acid p1 is selected from asparticacid or leucine, or is not present; and amino acid q1 is selected fromvaline, alanine, or threonine, or is not present; wherein CDR2bcomprises the amino acid sequence r1 s1 t1 u1 v1 w1 x1, wherein aminoacid r1 is selected from alanine, aspartic acid, leucine, tryptophan,glycine, or valine; amino acid s1 is selected from threonine, valine,glycine, or alanine; amino acid t1 is serine; amino acid u1 is selectedfrom serine, asparagine, or threonine; amino acid v1 is selected fromleucine, phenylalanine, or arginine; amino acid w1 is selected fromglutamine, alanine, or glutamic acid; and amino acid x1 is selected fromserine, arginine, or threonine; wherein CDR3b comprises the amino acidsequence y1 z1 a1′ b1′ c1′ d1′ e1′ f1′ g1′, wherein amino acid y1 isselected from glutamine, methionine, leucine, or histidine; amino acidz1 is selected from glutamine or lysine; amino acid a1′ is selected fromserine, threonine, alanine, histidine, tyrosine, or phenylalanine; aminoacid b1′ is selected from tyrosine, leucine, asparagine, or glycine;amino acid c1′ is selected from serine, glutamine, isoleucine, orlysine; amino acid d1′ is selected from threonine, phenylalanine,tyrosine, alanine, or serine; amino acid e1′ is proline; amino acid f1′is selected from leucine, phenylalanine, tryptophan, serine, orarginine; and amino acid g1′ is selected from threonine or serine; andwherein the polypeptide, in association with an antibody heavy chain,binds TR-2.

In certain embodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising at least two complementarity determining regions(CDR) selected from CDR1b, CDR2b, and CDR3b, wherein the polypeptide, inassociation with an antibody heavy chain, binds TR-2. In certainembodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising CDR1b, CDR2b, and CDR3b, wherein the polypeptide,in association with an antibody heavy chain, binds TR-2.

In certain embodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising an antibody light chain variable region. Incertain embodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising a human antibody light chain variable region. Incertain embodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising a light chain constant region. In certainembodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising a human light chain constant region. In certainembodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising an amino acid sequence as set forth in SEQ ID NO:36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ IDNO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64,SEQ ID NO: 66, or SEQ ID NO: 68. In certain embodiments, apolynucleotide comprises a sequence encoding a polypeptide comprising anon-human light chain constant region. In certain embodiments, apolynucleotide comprises a sequence encoding a polypeptide comprising alight chain constant region of a species other than human.

In certain embodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising at least one complementarity determining region(CDR) selected from amino acids 24 to 34 of SEQ ID NO: 36; amino acids50 to 56 of SEQ ID NO: 36; amino acids 89 to 97 of SEQ ID NO: 36; aminoacids 24 to 34 of SEQ ID NO: 38; amino acids 50 to 56 of SEQ ID NO: 38;amino acids 89 to 97 of SEQ ID NO: 38; amino acids 24 to 34 of SEQ IDNO: 40; amino acids 50 to 56 of SEQ ID NO: 40; amino acids 89 to 97 ofSEQ ID NO: 40; amino acids 24 to 34 of SEQ ID NO: 42; amino acids 50 to56 of SEQ ID NO: 42; amino acids 89 to 97 of SEQ ID NO: 42; amino acids24 to 34 of SEQ ID NO: 44; amino acids 50 to 56 of SEQ ID NO: 44; aminoacids 89 to 97 of SEQ ID NO: 44; amino acids 24 to 34 of SEQ ID NO: 46;amino acids 50 to 56 of SEQ ID NO: 46; amino acids 89 to 97 of SEQ IDNO: 46; amino acids 24 to 40 of SEQ ID NO: 48; amino acids 56 to 62 ofSEQ ID NO: 48; amino acids 95 to 103 of SEQ ID NO: 48; amino acids 24 to39 of SEQ ID NO: 50; amino acids 55 to 61 of SEQ ID NO: 50; amino acids94 to 102 of SEQ ID NO: 50; amino acids 24 to 40 of SEQ ID NO: 52; aminoacids 56 to 62 of SEQ ID NO: 52; amino acids 95 to 103 of SEQ ID NO: 52;amino acids 24 to 34 of SEQ ID NO: 54; amino acids 50 to 56 of SEQ IDNO: 54; amino acids 89 to 97 of SEQ ID NO: 54; amino acids 24 to 34 ofSEQ ID NO: 56; amino acids 50 to 56 of SEQ ID NO: 56; amino acids 89 to97 of SEQ ID NO: 56; amino acids 24 to 40 of SEQ ID NO: 58; amino acids56 to 62 of SEQ ID NO: 58; amino acids 95 to 103 of SEQ ID NO: 58; aminoacids 24 to 34 of SEQ ID NO: 60; amino acids 50 to 56 of SEQ ID NO: 60;amino acids 89 to 97 of SEQ ID NO: 60; amino acids 24 to 34 of SEQ IDNO: 62; amino acids 50 to 56 of SEQ ID NO: 62; amino acids 89 to 97 ofSEQ ID NO: 62; amino acids 24 to 35 of SEQ ID NO: 64; amino acids 51 to57 of SEQ ID NO: 64; amino acids 90 to 88 of SEQ ID NO: 64; amino acids24 to 34 of SEQ ID NO: 66; amino acids 50 to 57 of SEQ ID NO: 66; aminoacids 89 to 97 of SEQ ID NO: 66; amino acids 24 to 34 of SEQ ID NO: 68;amino acids 50 to 56 of SEQ ID NO: 68; and amino acids 89 to 97 of SEQID NO: 68, wherein the polypeptide, in association with an antibodyheavy chain, binds TR-2. In certain embodiments, a polynucleotidecomprises a sequence encoding a polypeptide comprising at least two ofthe CDRs of SEQ ID NOS. 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, or 68. In certain embodiments, a polynucleotide comprises asequence encoding a polypeptide comprising three of the CDRs of SEQ IDNOS. 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, or 68.

In certain embodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising amino acids 24 to 34 of SEQ ID NO: 36, aminoacids 50 to 56 of SEQ ID NO: 36, and amino acids 89-97 of SEQ ID NO: 36.In certain embodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising amino acids 24 to 34 of SEQ ID NO: 38, aminoacids 50 to 56 of SEQ ID NO: 38, and amino acids 89 to 97 of SEQ ID NO:38. In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising amino acids 24 to 34 of SEQ ID NO: 40,amino acids 50 to 56 of SEQ ID NO: 40, and amino acids 89 to 97 of SEQID NO: 40. In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising amino acids 24 to 34 of SEQ ID NO: 42,amino acids 50 to 56 of SEQ ID NO: 42, and amino acids 89 to 97 of SEQID NO: 42. In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising amino acids 24 to 34 of SEQ ID NO: 44,amino acids 50 to 56 of SEQ ID NO: 44, and amino acids 89-97 of SEQ IDNO: 44. In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising amino acids 24 to 34 of SEQ ID NO: 46,amino acids 50 to 56 of SEQ ID NO: 46, and amino acids 89 to 97 of SEQID NO: 46. In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising amino acids 24 to 40 of SEQ ID NO: 48,amino acids 56 to 62 of SEQ ID NO: 48, and amino acids 95 to 103 of SEQID NO: 48. In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising amino acids 24 to 39 of SEQ ID NO: 50,amino acids 55 to 61 of SEQ ID NO: 50, and amino acids 94 to 102 of SEQID NO: 50. In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising amino acids 24 to 40 of SEQ ID NO: 52,amino acids 56 to 62 of SEQ ID NO: 52, and amino acids 95 to 103 of SEQID NO: 52. In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising 24 to 34 of SEQ ID NO: 54, amino acids50 to 56 of SEQ ID NO: 54, and amino acids 89 to 97 of SEQ ID NO: 54. Incertain embodiments, a polynucleotide comprises a sequence encoding apolypeptide comprising amino acids 24 to 34 of SEQ ID NO: 56, aminoacids 50 to 56 of SEQ ID NO: 56, and amino acids 89 to 97 of SEQ ID NO:56, In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising amino acids 24 to 40 of SEQ ID NO: 58,amino acids 56 to 62 of SEQ ID NO: 58, and amino acids 95 to 103 of SEQID NO: 58. In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising amino acids 24 to 34 of SEQ ID NO: 60,amino acids 50 to 56 of SEQ ID NO: 60, and amino acids 89-97 of SEQ IDNO: 60. In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising amino acids 24 to 34 of SEQ ID NO: 62,amino acids 50 to 56 of SEQ ID NO: 62, and amino acids 89 to 97 of SEQID NO: 62. In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising amino acids 24 to 35 of SEQ ID NO: 64,amino acids 51 to 57 of SEQ ID NO: 64, and amino acids 90 to 88 of SEQID NO: 64. In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising amino acids 24 to 34 of SEQ ID NO: 66,amino acids 50 to 57 of SEQ ID NO: 66, and amino acids 89 to 97 of SEQID NO: 66. In certain embodiments, a polynucleotide comprises a sequenceencoding a polypeptide comprising amino acids 24 to 34 of SEQ ID NO: 68,amino acids 50 to 56 of SEQ ID NO: 68, and amino acids 89 to 97 of SEQID NO: 68.

In certain embodiments, this application discusses certainpolynucleotides encoding antibody heavy and light chains. In certainembodiments, this application discusses certain polynucleotides encodingan antibody heavy chain variable region. In certain embodiments, thisapplication discusses certain polynucleotides encoding a human antibodyheavy chain variable region. In certain embodiments, this applicationdiscusses certain polynucleotides encoding antibody light chain variableregions. In certain embodiments, this application discusses certainpolynucleotides encoding a human antibody light chain variable region.In certain embodiments, this application discusses certainpolynucleotides encoding an antibody heavy chain constant region. Incertain embodiments, this application discusses certain polynucleotidesencoding a human antibody heavy chain constant region. In certainembodiments, this application discusses certain polynucleotides encodingan antibody heavy chain constant region of a species other than human.In certain embodiments, this application discusses certainpolynucleotides encoding antibody light chain constant regions. Incertain embodiments, this application discusses certain polynucleotidesencoding a human antibody light chain constant region. In certainembodiments, this application discusses certain polynucleotides encodingan antibody light chain constant region of a species other than human.In certain embodiments, this application discusses certainpolynucleotides encoding a single-chain antibody.

In certain embodiments, these antibody heavy and light chainpolynucleotides and polypeptides are human antibody heavy and lightchain polynucleotides and polypeptides. In certain embodiments apolynucleotide comprises a nucleotide sequence as set forth in SEQ IDNOS. SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,or 67. In certain embodiments, a polynucleotide comprises a nucleotidesequence that has one or more deletions, additions, and/or substitutionsof one or more nucleotides of those sequences. In certain embodiments, apolynucleotide comprises a nucleotide sequence encoding an amino acidsequence comprising an amino acid sequence as set forth in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, or 68. Incertain embodiments, variable region sequences comprisingcomplementarity determining regions (CDRs), e.g., CDR1 through CDR3, areprovided. In certain embodiments, variable region polynucleotides andpolypeptides are human variable region polynucleotides and polypeptides.

The term “naturally occurring nucleotides” includes deoxyribonucleotidesand ribonucleotides. Deoxyribonucleotides include, but are not limitedto, adenosine, guanine, cytosine, and thymidine. Ribonucleotidesinclude, but are not limited to, adenosine, cytosine, thymidine, anduracil. The term “modified nucleotides” includes, but is not limited to,nucleotides with modified or substituted sugar groups and the like. Theterm “polynucleotide linkages” includes, but is not limited to,polynucleotide linkages such as phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phoshoraniladate, phosphoroamidate, and the like. See, e.g., LaPlancheet al. Nucl. Acids Res. 14:9081 (1986); Stec et al. J. Am. Chem. Soc.106:6077 (1984); Stein et al. Nucl. Acids Res. 16:3209 (1988); Zon etal. Anti-Cancer Drug Design 6:539 (1991); Zon et al. Oligonucleotidesand Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed.,Oxford University Press, Oxford England (1991)); Stec et al. U.S. Pat.No. 5,151,510; Uhlmann and Peyman Chemical Reviews 90:543 (1990). Incertain embodiments, a polynucleotide can include a label for detection.

The term “isolated polypeptide” refers to any polypeptide that (1) isfree of at least some proteins with which it would normally be found,(2) is essentially free of other proteins from the same source, e.g.,from the same species, (3) is expressed by a cell from a differentspecies, or (4) does not occur in nature.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein and refer to a polymer of two or more amino acidsjoined to each other by peptide bonds or modified peptide bonds, i.e.,peptide isosteres. The terms apply to amino acid polymers containingnaturally occurring amino acids as well as amino acid polymers in whichone or more amino acid residues is a non-naturally occurring amino acidor a chemical analogue of a naturally occurring amino acid. An aminoacid polymer may contain one or more amino acid residues that has beenmodified by one or more natural processes, such as post-translationalprocessing, and/or one or more amino acid residues that has beenmodified by one or more chemical modification techniques known in theart.

A “fragment” of a reference polypeptide refers to a contiguous stretchof amino acids from any portion of the reference polypeptide. A fragmentmay be of any length that is less than the length of the referencepolypeptide.

A “variant” of a reference polypeptide refers to a polypeptide havingone or more amino acid substitutions, deletions, or insertions relativeto the reference polypeptide. In certain embodiments, a variant of areference polypeptide has an altered post-translational modificationsite (i.e., a glycosylation site). In certain embodiments, both areference polypeptide and a variant of a reference polypeptide arespecific binding agents. In certain embodiments, both a referencepolypeptide and a variant of a reference polypeptide are antibodies.

Variants of a reference polypeptide include, but are not limited to,glycosylation variants. Glycosylation variants include variants in whichthe number and/or type of glycosylation sites have been altered ascompared to the reference polypeptide. In certain embodiments,glycosylation variants of a reference polypeptide comprise a greater ora lesser number of N-linked glycosylation sites than the referencepolypeptide. In certain embodiments, an N-linked glycosylation site ischaracterized by the sequence Asn-X-Ser or Asn-X-Thr, wherein the aminoacid residue designated as X may be any amino acid residue exceptproline. In certain embodiments, glycosylation variants of a referencepolypeptide comprise a rearrangement of N-linked carbohydrate chainswherein one or more N-linked glycosylation sites (typically those thatare naturally occurring) are eliminated and one or more new N-linkedsites are created.

Variants of a reference polypeptide include, but are not limited to,cysteine variants. In certain embodiments, cysteine variants includevariants in which one or more cysteine residues of the referencepolypeptide are replaced by one or more non-cysteine residues; and/orone or more non-cysteine residues of the reference polypeptide arereplaced by one or more cysteine residues. Cysteine variants may beuseful, in certain embodiments, when a particular polypeptide must berefolded into a biologically active conformation, e.g., after theisolation of insoluble inclusion bodies. In certain embodiments,cysteine variants of a reference polypeptide have fewer cysteineresidues than the reference polypeptide. In certain embodiments,cysteine variants of a reference polypeptide have an even number ofcysteines to minimize interactions resulting from unpaired cysteines. Incertain embodiments, cysteine variants have more cysteine residues thanthe native protein.

A “derivative” of a reference polypeptide refers to: a polypeptide: (1)having one or more modifications of one or more amino acid residues ofthe reference polypeptide; and/or (2) in which one or more peptidyllinkages has been replaced with one or more non-peptidyl linkages;and/or (3) in which the N-terminus and/or the C-terminus has beenmodified. Certain exemplary modifications include, but are not limitedto, acetylation, acylation, ADP-ribosylation, amidation, biotinylation,covalent attachment of flavin, covalent attachment of a heme moiety,covalent attachment of a nucleotide or nucleotide derivative, covalentattachment of a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination. In certain embodiments, both areference polypeptide and a derivative of a reference polypeptide arespecific binding agents. In certain embodiments, both a referencepolypeptide and a derivative of a reference polypeptide are antibodies.

Polypeptides include, but are not limited to, amino acid sequencesmodified either by natural processes, such as post-translationalprocessing, or by chemical modification techniques that are well knownin the art. In certain embodiments, modifications may occur anywhere ina polypeptide, including the peptide backbone, the amino acidside-chains and the amino or carboxyl termini. In certain suchembodiments, the modifications may be present to the same or varyingdegrees at several sites in a given polypeptide. In certain embodiments,a given polypeptide contains many types of modifications such asdeletions, additions, and/or substitutions of one or more amino acids ofa native sequence. In certain embodiments, polypeptides may be branchedand/or cyclic. Cyclic, branched and branched cyclic polypeptides mayresult from post-translational natural processes (including, but notlimited to, ubiquitination) or may be made by synthetic methods. Theterm “polypeptide” also encompasses sequences that comprise the aminoacid sequences of the heavy chain and/or light chain of an antibodyselected from Ab A, Ab B, Ab C, Ab D, Ab E, Ab F, Ab G, Ab H, Ab I, AbJ, Ab K, Ab L, Ab M, Ab N, Ab O, Ab P, and Ab Q, as described below (seeSEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, and68). The term “polypeptide” also encompasses sequences that have one ormore deletions, additions, and/or substitutions of one or more aminoacids of those sequences. In certain embodiments, certain polypeptidesequences comprise at least one complementarity determining region(CDR).

In certain embodiments, a polypeptide comprises at least onecomplementarity determining region (CDR) selected from CDR1a, CDR2a, andCDR3a wherein CDR1a comprises the amino acid sequence a b c d e f g h ij k l, wherein amino acid a is glycine, amino acid b is selected fromglycine, tyrosine, or phenylalanine; amino acid c is selected fromserine or threonine; amino acid d is selected from isoleucine orphenylalanine; amino acid e is selected from serine, threonine, orasparagine; amino acid f is selected from serine, aspartic acid,tyrosine, asparagine, threonine, or glycine; amino acid g is selectedfrom glycine, aspartic acid, or tyrosine; amino acid h is selected fromglycine, aspartic acid, tyrosine, asparagine, or serine; amino acid i isselected from tyrosine, isoleucine, histidine, methionine, ortryptophan; amino acid j is selected from asparagine, tyrosine,histidine, serine, or phenylalanine; amino acid k is tryptophan or isnot present; and amino acid l is serine or is not present; wherein CDR2acomprises the amino acid sequence m n o p q r s t u v w x y z a′ b′ c′,wherein amino acid m is selected from tryptophan, tyrosine, histidine,valine, glutamic acid, or serine; amino acid n is selected frommethionine or isoleucine; amino acid o is selected from asparagine,tyrosine, serine, tryptophan, or histidine; amino acid p is selectedfrom proline, tyrosine, serine, arginine, histidine, or asparagine;amino acid q is selected from asparagine, serine, or aspartic acid;amino acid r is selected from serine or glycine; amino acid s isselected from aspartic acid, serine, threonine, or arginine; amino acidt is selected from asparagine, threonine, alanine, isoleucine, ortyrosine; amino acid u is selected from threonine, tyrosine, leucine,lysine, asparagine, or isoleucine; amino acid v is selected fromglycine, tyrosine, aspartic acid, or cysteine; amino acid w is selectedfrom tyrosine- or asparagine; amino acid x is selected from alanine orproline; amino acid y is selected from glutamine, serine, or asparticacid; amino acid z is selected from lysine, leucine, or serine; aminoacid a′ is selected from phenylalanine, lysine, or valine; amino acid b′is selected from glutamine, serine, or lysine; and amino acid c′ isglycine or is not present; wherein CDR3a comprises the amino acidsequence d′ e′ f′ g′ h′ i′ j′ k′ l′ m′ n′ o′ p′ q′ r′ s′ t′ u′ v′ w′,wherein amino acid d′ is selected from tryptophan, aspartic acid,glycine, serine, or glutamic acid; amino acid e′ is selected fromasparagine, aspartic acid, glycine, arginine, serine, valine, orleucine; amino acid f′ is selected from histidine, serine, alanine,tyrosine, proline, asparagine, glycine or threonine; amino acid g′ isselected from tyrosine, serine, alanine, arginine, tryptophan, glycineor valine; amino acid h′ is selected from glycine, alanine, serine,asparagine, methionine, tyrosine, tryptophan, cysteine, or asparticacid; amino acid i′ is selected from serine, tryptophan, glycine,phenylalanine, aspartic acid, tyrosine, or threonine; amino acid j′ isselected from glycine, threonine, serine, leucine, valine, asparagine,tryptophan, or tyrosine; amino acid k′ is selected from serine,phenylalanine, aspartic acid, tryptophan, glycine, or tyrosine, or isnot present; amino acid l′ is selected from histidine, aspartic acid,alanine, tryptophan, tyrosine, serine, phenylalanine, valine, orglycine, or is not present; amino acid m′ is selected fromphenylalanine, tyrosine, glutamic acid, proline, aspartic acid,cysteine, isoleucine, or methionine, or is not present; amino acid n′ isselected from aspartic acid, phenylalanine, alanine, leucine, or serine,or is not present; amino acid o′ is selected from tyrosine, leucine,aspartic acid, phenylalanine, proline, or valine, or is not present;amino acid p′ is selected from leucine, aspartic acid, or tyrosine, oris not present; amino acid q′ is selected from serine or tyrosine, or isnot present; amino acid r′ is tyrosine or is not present; amino acid s′is selected from glycine or tyrosine, or is not present; amino acid t′is selected from glycine or methionine, or is not present; amino acid u′is selected from methionine or aspartic acid, or is not present; aminoacid v′ is selected from aspartic acid or valine, or is not present; andamino acid w′ is valine or is not present; and wherein the polypeptide,in association with an antibody light chain, binds TR-2.

In certain embodiments, a polypeptide comprises at least twocomplementarity determining regions (CDR) selected from CDR1a, CDR2a,and CDR3a, wherein the polypeptide, in association with an antibodylight chain, binds TR-2. In certain embodiments, a polypeptide comprisesCDR1a, CDR2a, and CDR3a, wherein the polypeptide, in association with anantibody light chain, binds TR-2.

In certain embodiments, a polypeptide comprises an antibody heavy chainvariable region. In certain embodiments, a polypeptide comprises a humanantibody heavy chain variable region. In certain embodiments, apolypeptide comprises a heavy chain constant region. In certainembodiments, a polypeptide comprises a human heavy chain constantregion. In certain embodiments, a polypeptide comprises an amino acidsequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16,SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, or SEQ ID NO: 34. Incertain embodiments, a polypeptide comprises a non-human heavy chainconstant region. In certain embodiments, a polypeptide comprises a heavychain constant region of a species other than human.

In certain embodiments, a polypeptide comprises at least onecomplementarity determining region (CDR) selected from amino acids 26 to35 of SEQ ID NO: 2; amino acids 50 to 66 of SEQ ID NO: 2; amino acids 99to 110 of SEQ ID NO: 2; amino acids 26 to 37 of SEQ ID NO: 4; aminoacids 52 to 67 of SEQ ID NO: 4; amino acids 100 to 109 of SEQ ID NO: 4;amino acids 26 to 37 of SEQ ID NO: 6; amino acids 52 to 67 of SEQ ID NO:6; amino acids 100 to 109 of SEQ ID NO: 6; amino acids 26 to 37 of SEQID NO: 8; amino acids 52 to 67 of SEQ ID NO: 8; amino acids 100 to 109of SEQ ID NO: 8; amino acids 26 to 35 of SEQ ID NO: 10, amino acids 50to 66 of SEQ ID NO: 10; amino acids 99-110 of SEQ ID NO: 10; amino acids26 to 35 of SEQ ID NO: 12; amino acids 50 to 66 of SEQ ID NO: 12; aminoacids 99-111 of SEQ ID NO: 12; amino acids 26 to 35 of SEQ ID NO: 14;amino acids 50 to 65 of SEQ ID NO: 14; amino acids 98 to 111 of SEQ IDNO: 14; amino acids 26 to 37 of SEQ ID NO: 16; amino acids 52 to 67 ofSEQ ID NO: 16; amino acids 100 to 109 of SEQ ID NO: 16; amino acids 26to 35 of SEQ ID NO: 18; amino acids 50 to 66 of SEQ ID NO: 18; aminoacids 99 to 105 of SEQ ID NO: 18; amino acids 26 to 35 of SEQ ID NO: 20;amino acids 50 to 66 of SEQ ID NO: 20; amino acids 99 to 118 of SEQ IDNO: 20; amino acids 26 to 35 of SEQ ID NO: 22; amino acids 50 to 66 ofSEQ ID NO: 22; amino acids 99 to 118 of SEQ ID NO: 22; amino acids 26 to35 of SEQ ID NO: 24; amino acids 50 to 65 of SEQ ID NO: 24; amino acids98 to 108 of SEQ ID NO: 24; amino acids 26 to 35 of SEQ ID NO: 26; aminoacids 50 to 66 of SEQ ID NO: 26; amino acids 99 to 110 of SEQ ID NO: 26;amino acids 26 to 35 of SEQ ID NO: 28; amino acids 50 to 66 of SEQ IDNO: 28; amino acids 99 to 117 of SEQ ID NO: 28; amino acids 26 to 37 ofSEQ ID NO: 30; amino acids 52 to 67 of SEQ ID NO: 30; amino acids 100 to111 of SEQ ID NO: 30; amino acids 26 to 37 of SEQ ID NO: 32; amino acids52 to 67 of SEQ ID NO: 32; amino acids 100 to 111 of SEQ ID NO: 32;amino acids 26 to 37 of SEQ ID NO: 34; amino acids 52 to 67 of SEQ IDNO: 34; and amino acids 100 to 111 of SEQ ID NO: 34, wherein thepolypeptide, in association with an antibody light chain, binds TR-2. Incertain embodiments, a polypeptide comprises at least two of the CDRs ofSEQ ID NOS. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,or 34. In certain embodiments, a polypeptide comprises at least three ofthe CDRs of SEQ ID NOS. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, or 34.

In certain embodiments, a polypeptide comprises amino acids 26 to 35 ofSEQ ID NO: 2, amino acids 50 to 66 of SEQ ID NO: 2, and amino acids 99to 110 of SEQ ID NO: 2. In certain embodiments, a polypeptide comprisesamino acids 26 to 37 of SEQ ID NO: 4, amino acids 52 to 67 of SEQ ID NO:4, and amino acids 100 to 109 of SEQ ID NO: 4. In certain embodiments, apolypeptide comprises amino acids 26 to 37 of SEQ ID NO: 6, amino acids52 to 67 of SEQ ID NO: 6, and amino acids 100 to 109 of SEQ ID NO: 6. Incertain embodiments, a polypeptide comprises amino acids 26 to 37 of SEQID NO: 8, amino acids 52 to 67 of SEQ ID NO: 8, and amino acids 100 to109 of SEQ ID NO: 8. In certain embodiments, a polypeptide comprisesamino acids 26 to 35 of SEQ ID NO: 10, amino acids 50 to 66 of SEQ IDNO:10, and amino acids 99-110 of SEQ ID NO:10. In certain embodiments, apolypeptide comprises amino acids 26 to 35 of SEQ ID NO: 12, amino acids50 to 66 of SEQ ID NO: 12, and amino acids 99-111 of SEQ ID NO: 12. Incertain embodiments, a polypeptide comprises amino acids 26 to 35 of SEQID NO: 14, amino acids 50 to 65 of SEQ ID NO: 14, and amino acids 98 to111 of SEQ ID NO: 14. In certain embodiments, a polypeptide comprisesamino acids 26 to 37 of SEQ ID NO: 16, amino acids 52 to 67 of SEQ IDNO: 16, and amino acids 100 to 109 of SEQ ID NO: 16. In certainembodiments, a polypeptide comprises amino acids 26 to 35 of SEQ ID NO:18, amino acids 50 to 66 of SEQ ID NO: 18, and amino acids 99 to 105 ofSEQ ID NO: 18. In certain embodiments, a polypeptide comprises aminoacids 26 to 35 of SEQ ID NO: 20, amino acids 50 to 66 of SEQ ID NO: 20,and amino acids 99 to 118 of SEQ ID NO: 20. In certain embodiments, apolypeptide comprises amino acids 26 to 35 of SEQ ID NO: 22, amino acids50 to 66 of SEQ ID NO: 22, and amino acids 99 to 118 of SEQ ID NO: 22.In certain embodiments, a polypeptide comprises amino acids 26 to 35 ofSEQ ID NO: 24, amino acids 50 to 65 of SEQ ID NO: 24, and amino acids 98to 108 of SEQ ID NO: 24. In certain embodiments, a polypeptide comprisesamino acids 26 to 35 of SEQ ID NO: 26, amino acids 50 to 66 of SEQ IDNO: 26, and amino acids 99 to 110 of SEQ ID NO: 26. In certainembodiments, a polypeptide comprises amino acids 26 to 35 of SEQ ID NO:28, amino acids 50 to 66 of SEQ ID NO: 28, and amino acids 99 to 117 ofSEQ ID NO: 28. In certain embodiments, a polypeptide comprises aminoacids 26 to 37 of SEQ ID NO: 30, amino acids 52 to 67 of SEQ ID NO: 30,and amino acids 100 to 111 of SEQ ID NO: 30. In certain embodiments, apolypeptide comprises amino acids 26 to 37 of SEQ ID NO: 32, amino acids52 to 67 of SEQ ID NO: 32, and amino acids 100 to 111 of SEQ ID NO: 32.In certain embodiments, a polypeptide comprises amino acids 26 to 37 ofSEQ ID NO: 34, amino acids 52 to 67 of SEQ ID NO: 34, and amino acids100 to 111 of SEQ ID NO: 34.

In certain embodiments, a polypeptide comprises at least onecomplementarity determining region (CDR) selected from CDR1b, CDR2b, andCDR3b, wherein CDR1b comprises a1 b1 c1 d1 e1 f1 g1 h1 i1 j1 k1 l1 m1 n1o1 p1 q1, wherein amino acid a1 is selected from arginine or lysine;amino acid b1 is selected from threonine, alanine, or serine; amino acidc1 is serine; amino acid d1 is glutamine; amino acid e1 is selected fromserine or glycine; amino acid f1 is selected from isoleucine, leucine,or valine; amino acid g1 is selected from serine, leucine, or arginine;amino acid h1 is selected from threonine, serine, isoleucine,asparagine, arginine, histidine, or tyrosine; amino acid i1 is selectedfrom tyrosine, arginine, tryptophan, aspartic acid, or serine; j1 isselected from leucine, isoleucine, asparagine, tyrosine, or serine;amino acid k1 is selected from asparagine, glycine, valine, alanine, orleucine; amino acid l1 is selected from tyrosine, alanine, orasparagine, or is not present; amino acid m1 is selected from asparagineor lysine, or is not present; amino acid n1 is selected from tyrosine,asparagine, or isoleucine, or is not present; amino acid o1 is selectedfrom leucine or tyrosine, or is not present; amino acid p1 is selectedfrom aspartic acid or leucine, or is not present; and amino acid q1 isselected from valine, alanine, or threonine, or is not present; whereinCDR2b comprises the amino acid r1 s1 t1 u1 v1 w1 x1, wherein amino acidr1 is selected from alanine, aspartic acid, leucine, tryptophan,glycine, or valine; amino acid s1 is selected from threonine, valine,glycine, or alanine; amino acid t1 is serine; amino acid u1 is selectedfrom serine, asparagine, or threonine; amino acid v1 is selected fromleucine, phenylalanine, or arginine; amino acid w1 is selected fromglutamine, alanine, or glutamic acid; and amino acid x1 is selected fromserine, arginine, or threonine; wherein CDR3b comprises the amino acidsequence y1 z1 a1′ b1′ c1′ d1′ e1′ f1′ g1′, wherein amino acid y1 isselected from glutamine, methionine, leucine, or histidine; amino acidz1 is selected from glutamine or lysine; amino acid a1′ is selected fromserine, threonine, alanine, histidine, tyrosine, or phenylalanine; aminoacid b1′ is selected from tyrosine, leucine, asparagine, or glycine;amino acid c1′ is selected from serine, glutamine, isoleucine, orlysine; amino acid d1′ is selected from threonine, phenylalanine,tyrosine, alanine, or serine; amino acid e1′ is proline; amino acid f1′is selected from leucine, phenylalanine, tryptophan, serine, orarginine; and amino acid g1′ is selected from threonine or serine; andwherein the polypeptide, in association with an antibody heavy chain,binds TR-2.

In certain embodiments, a polypeptide comprises at least twocomplementarity determining regions (CDR) selected from CDR1b, CDR2b,and CDR3b, wherein the polypeptide, in association with an antibodyheavy chain, binds TR-2. In certain embodiments, a polypeptide comprisesCDR1b, CDR2b, and CDR3b, wherein the polypeptide, in association with anantibody heavy chain, binds TR-2.

In certain embodiments, a polypeptide comprises an antibody light chainvariable region. In certain embodiments, a polypeptide comprises a humanantibody light chain variable region. In certain embodiments, apolypeptide comprises a light chain constant region. In certainembodiments, a polypeptide comprises a human light chain constantregion. In certain embodiments, a polypeptide comprises an amino acidsequence as set forth in SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO:50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ IDNO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, or SEQ ID NO: 68.In certain embodiments, a polypeptide comprises a non-human light chainconstant region. In certain embodiments, a polypeptide comprises a lightchain constant region of a species other than human.

In certain embodiments, a polypeptide which comprises at least onecomplementarity determining region (CDR) selected from amino acids 24 to34 of SEQ ID NO: 36; amino acids 50 to 56 of SEQ ID NO: 36; amino acids89-97 of SEQ ID NO: 36; amino acids 24 to 34 of SEQ ID NO: 38; aminoacids 50 to 56 of SEQ ID NO: 38; amino acids 89 to 97 of SEQ ID NO: 38;amino acids 24 to 34 of SEQ ID NO: 40; amino acids 50 to 56 of SEQ IDNO: 40; amino acids 89 to 97 of SEQ ID NO: 40; amino acids 24 to 34 ofSEQ ID NO: 42; amino acids 50 to 56 of SEQ ID NO: 42; amino acids 89 to97 of SEQ ID NO: 42; amino acids 24 to 34 of SEQ ID NO: 44; amino acids50 to 56 of SEQ ID NO: 44; amino acids 89-97 of SEQ ID NO: 44; aminoacids 24 to 34 of SEQ ID NO: 46; amino acids 50 to 56 of SEQ ID NO: 46;amino acids 89 to 97 of SEQ ID NO: 46; amino acids 24 to 40 of SEQ IDNO: 48; amino acids 56 to 62 of SEQ ID NO: 48; amino acids 95 to 103 ofSEQ ID NO: 48; amino acids 24 to 39 of SEQ ID NO: 50; amino acids 55 to61 of SEQ ID NO: 50; amino acids 94 to 102 of SEQ ID NO: 50; amino acids24 to 40 of SEQ ID NO: 52; amino acids 56 to 62 of SEQ ID NO: 52; aminoacids 95 to 103 of SEQ ID NO: 52; 24 to 34 of SEQ ID NO: 54; amino acids50 to 56 of SEQ ID NO: 54; amino acids 89 to 97 of SEQ ID NO: 54; aminoacids 24 to 34 of SEQ ID NO: 56, amino acids 50 to 56 of SEQ ID NO: 56;amino acids 89 to 97 of SEQ ID NO: 56; amino acids 24 to 40 of SEQ IDNO: 58; amino acids 56 to 62 of SEQ ID NO: 58; amino acids 95 to 103 ofSEQ ID NO: 58; amino acids 24 to 34 of SEQ ID NO: 60; amino acids 50 to56 of SEQ ID NO: 60; amino acids 89-97 of SEQ ID NO: 60; amino acids 24to 34 of SEQ ID NO: 62; amino acids 50 to 56 of SEQ ID NO: 62; aminoacids 89 to 97 of SEQ ID NO: 62; amino acids 24 to 35 of SEQ ID NO: 64;amino acids 51 to 57 of SEQ ID NO: 64; amino acids 90 to 88 of SEQ IDNO: 64; amino acids 24 to 34 of SEQ ID NO: 66; amino acids 50 to 57 ofSEQ ID NO: 66; amino acids 89 to 97 of SEQ ID NO: 66; amino acids 24 to34 of SEQ ID NO: 68; amino acids 50 to 56 of SEQ ID NO: 68; and aminoacids 89 to 97 of SEQ ID NO: 68, wherein the polypeptide, in associationwith an antibody heavy chain, binds TR-2. In certain embodiments, apolypeptide comprises at least two of the CDRs of SEQ ID NOS. 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, or 68. Incertain embodiments, a polypeptide comprises at least three of the CDRsof SEQ ID NOS. 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, or 68.

In certain embodiments, a polypeptide comprises amino acids 24 to 34 ofSEQ ID NO: 36, amino acids 50 to 56 of SEQ ID NO: 36, and amino acids89-97 of SEQ ID NO: 36. In certain embodiments, a polypeptide comprisesamino acids 24 to 34 of SEQ ID NO: 38, amino acids 50 to 56 of SEQ IDNO: 38, and amino acids 89 to 97 of SEQ ID NO: 38. In certainembodiments, a polypeptide comprises amino acids 24 to 34 of SEQ ID NO:40, amino acids 50 to 56 of SEQ ID NO: 40, and amino acids 89 to 97 ofSEQ ID NO: 40. In certain embodiments, a polypeptide comprises aminoacids 24 to 34 of SEQ ID NO: 42, amino acids 50 to 56 of SEQ ID NO: 42,and amino acids 89 to 97 of SEQ ID NO: 42. In certain embodiments, apolypeptide comprises amino acids 24 to 34 of SEQ ID NO: 44, amino acids50 to 56 of SEQ ID NO: 44, and amino acids 89-97 of SEQ ID NO: 44. Incertain embodiments, a polypeptide comprises amino acids 24 to 34 of SEQID NO: 46, amino acids 50 to 56 of SEQ ID NO: 46, and amino acids 89 to97 of SEQ ID NO: 46. In certain embodiments, a polypeptide comprisesamino acids 24 to 40 of SEQ ID NO: 48, amino acids 56 to 62 of SEQ IDNO: 48, and amino acids 95 to 103 of SEQ ID NO: 48. In certainembodiments, a polypeptide comprises amino acids 24 to 39 of SEQ ID NO:50, amino acids 55 to 61 of SEQ ID NO: 50, and amino acids 94 to 102 ofSEQ ID NO: 50. In certain embodiments, a polypeptide comprises aminoacids 24 to 40 of SEQ ID NO: 52, amino acids 56 to 62 of SEQ ID NO: 52,and amino acids 95 to 103 of SEQ ID NO: 52. In certain embodiments, apolypeptide comprises amino acids 24 to 34 of SEQ ID NO: 54, amino acids50 to 56 of SEQ ID NO: 54, and amino acids 89 to 97 of SEQ ID NO: 54. Incertain embodiments, a polypeptide comprises amino acids 24 to 34 of SEQID NO: 56, amino acids 50 to 56 of SEQ ID NO: 56, and amino acids 89 to97 of SEQ ID NO: 56, In certain embodiments, a polypeptide comprisesamino acids 24 to 40 of SEQ ID NO: 58, amino acids 56 to 62 of SEQ IDNO: 58, and amino acids 95 to 103 of SEQ ID NO: 58. In certainembodiments, a polypeptide comprises amino acids 24 to 34 of SEQ ID NO:60, amino acids 50 to 56 of SEQ ID NO: 60, and amino acids 89-97 of SEQID NO: 60. In certain embodiments, a polypeptide comprises amino acids24 to 34 of SEQ ID NO: 62, amino acids 50 to 56 of SEQ ID NO: 62, andamino acids 89 to 97 of SEQ ID NO: 62. In certain embodiments, apolypeptide comprises amino acids 24 to 35 of SEQ ID NO: 64, amino acids51 to 57 of SEQ ID NO: 64, and amino acids 90 to 88 of SEQ ID NO: 64. Incertain embodiments, a polypeptide comprises amino acids 24 to 34 of SEQID NO: 66, amino acids 50 to 57 of SEQ ID NO: 66, and amino acids 89 to97 of SEQ ID NO: 66. In certain embodiments, a polypeptide comprisesamino acids 24 to 34 of SEQ ID NO: 68, amino acids 50 to 56 of SEQ IDNO: 68, and amino acids 89 to 97 of SEQ ID NO: 68.

The term “naturally-occurring” as applied to an object means that anobject can be found in nature. For example, a polypeptide orpolynucleotide that is present in an organism (including viruses) thatcan be isolated from a source in nature and which has not beenintentionally modified by man in the laboratory or otherwise isnaturally-occurring.

The term “operably linked” as used herein refers to components that arein a relationship permitting them to function in their intended manner.For example, in the context of a polynucleotide sequence, a controlsequence may be “operably linked” to a coding sequence when the controlsequence and coding sequence are in association with each other in sucha way that expression of the coding sequence is achieved underconditions compatible with the functioning of the control sequence.

The term “control sequence” refers to polynucleotide sequences which mayeffect the expression and processing of coding sequences with which theyare in association. The nature of such control sequences may differdepending upon the host organism. Certain exemplary control sequencesfor prokaryotes include, but are not limited to, promoters, ribosomalbinding sites, and transcription termination sequences. Certainexemplary control sequences for eukaryotes include, but are not limitedto, promoters, enhancers, and transcription termination sequences. Incertain embodiments, “control sequences” can include leader sequencesand/or fusion partner sequences.

In certain embodiments, a first polynucleotide coding sequence isoperably linked to a second polynucleotide coding sequence when thefirst and second polynucleotide coding sequences are transcribed into asingle contiguous mRNA that can be translated into a single contiguouspolypeptide.

In the context of polypeptides, two or more polypeptides are “operablylinked” if each linked polypeptide is able to function in its intendedmanner. A polypeptide that is able to function in its intended mannerwhen operably linked to another polypeptide may or may not be able tofunction in its intended manner when not operably linked to anotherpolypeptide. For example, in certain embodiments, a first polypeptidemay be unable to function in its intended manner when unlinked, but maybe stabilized by being linked to a second polypeptide such that itbecomes able to function in its intended manner. Alternatively, incertain embodiments, a first polypeptide may be able to function in itsintended manner when unlinked, and may retain that ability when operablylinked to a second polypeptide.

As used herein, two or more polypeptides are “fused” when the two ormore polypeptides are linked by translating them as a single contiguouspolypeptide sequence or by synthesizing them as a single contiguouspolypeptide sequence. In certain embodiments, two or more fusedpolypeptides may have been translated in vivo from two or more operablylinked polynucleotide coding sequences. In certain embodiments, two ormore fused polypeptides may have been translated in vitro from two ormore operably linked polynucleotide coding sequences.

As used herein, two or more polypeptides are “operably fused” if eachlinked polypeptide is able to function in its intended manner.

In certain embodiments, a first polypeptide that contains two or moredistinct polypeptide units is considered to be linked to a secondpolypeptide so long as at least one of the distinct polypeptide units ofthe first polypeptide is linked to the second polypeptide. As anon-limiting example, in certain embodiments, an antibody is consideredlinked to a second polypeptide in all of the following instances: (a)the second polypeptide is linked to one of the heavy chain polypeptidesof the antibody; (b) the second polypeptide is linked to one of thelight chain polypeptides of the antibody; (c) a first molecule of thesecond polypeptide is linked to one of the heavy chain polypeptides ofthe antibody and a second molecule of the second polypeptide is linkedto one of the light chain polypeptides of the antibody; and (d) firstand second molecules of the second polypeptide are linked to the firstand second heavy chain polypeptides of the antibody and third and fourthmolecules of the second polypeptide are linked to first and second lightchain polypeptides of the antibody.

In certain embodiments, the language “a first polypeptide linked to asecond polypeptide” encompasses situations where: (a) only one moleculeof a first polypeptide is linked to only one molecule of a secondpolypeptide; (b) only one molecule of a first polypeptide is linked tomore than one molecule of a second polypeptide; (c) more than onemolecule of a first polypeptide is linked to only one molecule of asecond polypeptide; and (d) more than one molecule of a firstpolypeptide is linked to more than one molecule of a second polypeptide.In certain embodiments, when a linked molecule comprises more than onemolecule of a first polypeptide and only one molecule of a secondpolypeptide, all or fewer than all of the molecules of the firstpolypeptide may be covalently or noncovalently linked to the secondpolypeptide. In certain embodiments, when a linked molecule comprisesmore than one molecule of a first polypeptide, one or more molecules ofthe first polypeptide may be covalently or noncovalently linked to othermolecules of the first polypeptide.

As used herein, a “flexible linker” refers to any linker that is notpredicted, according to its chemical structure, to be fixed inthree-dimensional space. One skilled in the art can predict whether aparticular linker is flexible in its intended context. In certainembodiments, a peptide linker comprising 3 or more amino acids is aflexible linker.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)). In certain embodiments, one or moreunconventional amino acids may be incorporated into a polypeptide. Theterm “unconventional amino acid” refers to any amino acid that is notone of the twenty conventional amino acids. The term “non-naturallyoccurring amino acids” refers to amino acids that are not found innature. Non-naturally occurring amino acids are a subset ofunconventional amino acids. Unconventional amino acids include, but arenot limited to, stereoisomers (e.g., D-amino acids) of the twentyconventional amino acids, unnatural amino acids such as α-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid,homoserine, homocysteine, 4-hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,σ-N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline) known in the art. In the polypeptide notation usedherein, the left-hand direction is the amino terminal direction and theright-hand direction is the carboxy-terminal direction, in accordancewith standard usage and convention.

In certain embodiments, conservative amino acid substitutions includesubstitution with one or more unconventional amino acid residues. Incertain embodiments, unconventional amino acid residues are incorporatedby chemical peptide synthesis rather than by synthesis in biologicalsystems.

The term “acidic residue” refers to an amino acid residue in D- orL-form that comprises at least one acidic group when incorporated into apolypeptide between two other amino acid residues that are the same ordifferent. In certain embodiments, an acidic residue comprises asidechain that comprises at least one acidic group. Exemplary acidicresidues include, but are not limited to, aspartic acid (D) and glutamicacid (E). In certain embodiments, an acidic residue may be anunconventional amino acid.

The term “aromatic residue” refers to an amino acid residue in D- orL-form that comprises at least one aromatic group. In certainembodiments, an aromatic residue comprises a sidechain that comprises atleast one aromatic group. Exemplary aromatic residues include, but arenot limited to, phenylalanine (F), tyrosine (Y), and tryptophan (W). Incertain embodiments, an aromatic residue may be an unconventional aminoacid.

The term “basic residue” refers to an amino acid residue in F- or L-formthat may comprise at least one basic group when incorporated into apolypeptide next to one or more amino acid residues that are the same ordifferent. In certain embodiments, a basic residue comprises a sidechainthat comprises at least one basic group. Exemplary basic residuesinclude, but are not limited to, histidine (H), lysine (K), and arginine(R). In certain embodiments, a basic residue may be an unconventionalamino acid.

The term “neutral hydrophilic residue” refers to an amino acid residuein D- or L-form that comprises at least one hydrophilic and/or polargroup, but does not comprise an acidic or basic group when incorporatedinto a polypeptide next to one or more amino acid residues that are thesame or different. Exemplary neutral hydrophilic residues include, butare not limited to, alanine (A), cysteine (C), serine (S), threonine(T), asparagine (N), and glutamine (Q). In certain embodiments, aneutral hydrophilic residue may be an unconventional amino acid.

The terms “lipophilic residue” and “Laa” refer to an amino acid residuein D- or L-form having at least one uncharged, aliphatic and/or aromaticgroup. In certain embodiments, a lipophilic residue comprises a sidechain that comprises at least one uncharged, aliphatic, and/or aromaticgroup. Exemplary lipophilic sidechains include, but are not limited to,alanine (A), phenylalanine (F), isoleucine (I), leucine (L), norleucine(Nile), methionine (M), valine (V), tryptophan (W), and tyrosine (Y). Incertain embodiments, a lipophilic residue may be an unconventional aminoacid.

The term “amphiphilic residue” refers to an amino acid residue in D- orL-form that is capable of being either a hydrophilic or lipophilicresidue. An exemplary amphiphilic residue includes, but is not limitedto, alanine (A). In certain embodiments, an amphiphilic residue may bean unconventional amino acid.

The term “nonfunctional residue” refers to an amino acid residue in D-or L-form that lacks acidic, basic, and aromatic groups whenincorporated into a polypeptide next to one or more amino acid residuesthat are the same or different. Exemplary nonfunctional amino acidresidues include, but are not limited to, methionine (M), glycine (G),alanine (A), valine (V), isoleucine (I), leucine (L), and norleucine(Nle). In certain embodiments, a nonfunctional residue may be anunconventional amino acid.

In certain embodiments, glycine (G) and proline (P) are considered aminoacid residues that can influence polypeptide chain orientation.

In certain embodiments, a conservative substitution may involvereplacing a member of one residue type with a member of the same residuetype. As a non-limiting example, in certain embodiments, a conservativesubstitution may involve replacing an acidic residue, such as D, with adifferent acidic residue, such as E. In certain embodiments, anon-conservative substitution may involve replacing a member of oneresidue type with a member of a different residue type. As anon-limiting example, in certain embodiments, a non-conservativesubstitution may involve replacing an acidic residue, such as D, with abasic residue, such as K. In certain embodiments, a cysteine residue issubstituted with another amino acid residue to prevent disulfide bondformation with that position in the polypeptide.

In making conservative or non-conservative substitutions, according tocertain embodiments, the hydropathic index of amino acids may beconsidered. Each amino acid has been assigned a hydropathic index on thebasis of its hydrophobicity and charge characteristics. The hydropathicindices of the 20 naturally-occurring amino acids are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art.Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is known in certaininstances that certain amino acids may be substituted for other aminoacids having a similar hydropathic index or score and still retain asimilar biological activity. In making changes based upon thehydropathic index, in certain embodiments, the substitution of aminoacids whose hydropathic indices are within ±2 is included. In certainembodiments, those which are within ±1 are included, and in certainembodiments, those within ±0.5 are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional protein or peptidethereby created is intended for use in immunological embodiments, as inthe present case. In certain embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the polypeptide.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5)and tryptophan (−3.4). In making changes based upon similarhydrophilicity values, in certain embodiments, the substitution of aminoacids whose hydrophilicity values are within ±2 is included, in certainembodiments, those which are within ±1 are included, and in certainembodiments, those within ±0.5 are included. In certain instances, onemay also identify epitopes from primary amino acid sequences on thebasis of hydrophilicity. These regions are also referred to as “epitopiccore regions.”

Exemplary amino acid substitutions are set forth in Table 1.

TABLE 1 Amino Acid Substitutions More specific Original Exemplaryexemplary Residues Substitutions Substitutions Ala Val, Leu, Ile Val ArgLys, Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn AsnGlu Asp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val,Met, Ala, Leu Phe, Norleucine Leu Norleucine, Ile, Ile Val, Met, Ala,Phe Lys Arg, 1,4 Diamino-butyric Arg Acid, Gln, Asn Met Leu, Phe, IleLeu Phe Leu, Val, Ile, Ala, Leu Tyr Pro Ala Gly Ser Thr, Ala, Cys ThrThr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met,Leu, Phe, Leu Ala, Norleucine

Similarly, as used herein, unless specified otherwise, the left-hand endof single-stranded polynucleotide sequences is the 5′ end; the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to herein as the transcription direction;sequence regions on the DNA strand having the same sequence as the RNAand which are 5′ to the 5′ end of the RNA transcript are referred toherein as “upstream sequences”; sequence regions on the DNA strandhaving the same sequence as the RNA and which are 3′ to the 3′ end ofthe RNA transcript are referred to herein as “downstream sequences.”

In certain embodiments, conservative amino acid substitutions encompassnon-naturally occurring amino acid residues, which are typicallyincorporated by chemical peptide synthesis rather than by synthesis inbiological systems. Those non-naturally occurring amino acid residuesinclude, but are not limited to, peptidomimetics and other reversed orinverted forms of amino acid moieties.

A skilled artisan will be able to determine suitable substitutionvariants of a reference polypeptide as set forth herein using well-knowntechniques. In certain embodiments, one skilled in the art may identifysuitable areas of the molecule that may be changed without destroyingactivity by targeting regions not believed to be important for activity.In certain embodiments, one can identify residues and portions of themolecules that are conserved among similar polypeptides. In certainembodiments, even areas that may be important for biological activity,including, but not limited to, the CDRs of an antibody, or that may beimportant for structure may be subject to conservative amino acidsubstitutions without destroying the biological activity or withoutadversely affecting the polypeptide structure.

Additionally, in certain embodiments, one skilled in the art can reviewstructure-function studies identifying residues in similar polypeptidesthat are important for activity and/or structure. In view of such acomparison, in certain embodiments, one can predict the importance ofamino acid residues in a polypeptide that correspond to amino acidresidues which are important for activity or structure in similarpolypeptides. In certain embodiments, one skilled in the art may opt forchemically similar amino acid substitutions for such predicted importantamino acid residues.

In certain embodiments, one skilled in the art can also analyze thethree-dimensional structure and amino acid sequence in relation to thatstructure in similar polypeptides. In view of such information, oneskilled in the art may predict the alignment of amino acid residues ofan antibody with respect to its three dimensional structure. In certainembodiments, one skilled in the art may choose not to make radicalchanges to amino acid residues predicted to be on the surface of theprotein, since such residues may be involved in important interactionswith other molecules. Moreover, in certain embodiments, one skilled inthe art may generate test variants containing a single amino acidsubstitution at each desired amino acid residue. In certain embodiments,the variants can then be screened using activity assays known to thoseskilled in the art. For example, in certain embodiments, the variantscan be screened for their ability to bind to TR-2. In certainembodiments, such variants could be used to gather information aboutsuitable variants. For example, in certain embodiments, if onediscovered that a change to a particular amino acid residue resulted indestroyed, undesirably reduced, or unsuitable activity, variants withsuch a change may be avoided. In other words, based on informationgathered from such routine experiments, one skilled in the art canreadily determine the amino acids where further substitutions should beavoided, either alone or in combination with other mutations.

A number of scientific publications have been devoted to the predictionof secondary structure. See Moult J., Curr. Op. in Biotech.,7(4):422-427 (1996), Chou et al., Biochemistry, 13(2):222-245 (1974);Chou et al., Biochemistry, 113(2):211-222 (1974); Chou et al., Adv.Enzymol. Relat. Areas Mol. Biol., 47:45-148 (1978); Chou et al., Ann.Rev. Biochem., 47:251-276 and Chou et al., Biophys. J., 26:367-384(1979). Moreover, computer programs are currently available to assistwith predicting secondary structure. One method of predicting secondarystructure is based upon homology modeling. For example, two polypeptidesor proteins which have a sequence identity of greater than 30%, orsimilarity greater than 40% often have similar structural topologies.The recent growth of the protein structural database (PDB) has providedenhanced predictability of secondary structure, including the potentialnumber of folds within a polypeptide's or protein's structure. See Holmet al., Nucl. Acid. Res., 27(1):244-247 (1999). It has been suggestedthat there are a limited number of folds in a given polypeptide orprotein and that once a critical number of structures have beenresolved, structural prediction will become dramatically more accurate.See, e.g., Brenner et al., Curr. Op. Struct. Bio., 7(3):369-376 (1997).

Additional exemplary methods of predicting secondary structure include,but are not limited to, “threading” (Jones, D., Curr. Opin. Struct.Biol., 7(3):377-87 (1997); Sippl et al., Structure, 4(1):15-19 (1996)),“profile analysis” (Bowie et al., Science, 253:164-170 (1991); Gribskovet al., Meth. Enzym., 183:146-159 (1990); Gribskov et al., Proc. Nat.Acad. Sci., 84(13):4355-4358 (1987)), and “evolutionary linkage” (SeeHolm, supra (1999), and Brenner, supra (1997)).

In certain embodiments, the identity and similarity of relatedpolypeptides can be readily calculated by known methods. Such methodsinclude, but are not limited to, those described in ComputationalMolecular Biology, Lesk, A. M., ed., Oxford University Press, New York(1988); Biocomputing: Informatics and Genome Projects, Smith, D. W.,ed., Academic Press, New York (1993); Computer Analysis of SequenceData, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press,New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinje,G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. andDevereux, J., eds., M. Stockton Press, New York (1991); and Carillo etal., SIAM J. Applied Math., 48:1073 (1988). In certain embodiments,polypeptides have amino acid sequences that are about 90 percent, orabout 95 percent, or about 96 percent, or about 97 percent, or about 98percent, or about 99 percent identical to amino acid sequences shown inFIGS. 3-19.

In certain embodiments, methods to determine identity are designed togive the largest match between the sequences tested. In certainembodiments, certain methods to determine identity are described inpublicly available computer programs. Certain computer program methodsto determine identity between two sequences include, but are not limitedto, the GCG program package, including GAP (Devereux et al., Nucl. Acid.Res., 12:387 (1984); Genetics Computer Group, University of Wisconsin,Madison, Wis., BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol.Biol., 215:403-410 (1990)). The BLASTX program is publicly availablefrom the National Center for Biotechnology Information (NCBI) and othersources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894;Altschul et al., supra (1990)). In certain embodiments, the SmithWaterman algorithm, which is known in the art, may also be used todetermine identity.

Certain alignment schemes for aligning two amino acid sequences mayresult in the matching of only a short region of the two sequences, andthis small aligned region may have very high sequence identity eventhough there is no significant relationship between the two full-lengthsequences. Accordingly, in certain embodiments, the selected alignmentmethod (GAP program) will result in an alignment that spans at least 50contiguous amino acids of the target polypeptide.

For example, using the computer algorithm GAP (Genetics Computer Group,University of Wisconsin, Madison, Wis.), two polypeptides for which thepercent sequence identity is to be determined are aligned for optimalmatching of their respective amino acids (the “matched span”, asdetermined by the algorithm). In certain embodiments, a gap openingpenalty (which is calculated as 3× the average diagonal; the “averagediagonal” is the average of the diagonal of the comparison matrix beingused; the “diagonal” is the score or number assigned to each perfectamino acid match by the particular comparison matrix) and a gapextension penalty (which is usually 1/10 times the gap opening penalty),as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used inconjunction with the algorithm. In certain embodiments, a standardcomparison matrix is also used by the algorithm. See, e.g., Dayhoff etal., Atlas of Protein Sequence and Structure, 5(3)(1978) for the PAM 250comparison matrix; Henikoff et al., Proc. Natl. Acad. Sci. USA,89:10915-10919 (1992) for the BLOSUM 62 comparison matrix.

In certain embodiments, the parameters for a polypeptide sequencecomparison include the following:

Algorithm: Needleman et al., J. Mol. Biol., 48:443-453 (1970);

Comparison matrix: BLOSUM 62 from Henikoff et al., supra (1992);

Gap Penalty: 12

Gap Length Penalty: 4

Threshold of Similarity: 0

In certain embodiments, the GAP program may be useful with the aboveparameters. In certain embodiments, the aforementioned parameters arethe default parameters for polypeptide comparisons (along with nopenalty for end gaps) using the GAP algorithm.

According to certain embodiments, amino acid substitutions are thosewhich: (1) reduce susceptibility to proteolysis, (2) reducesusceptibility to oxidation, (3) alter binding affinity for formingprotein complexes, (4) alter binding affinities, and/or (4) confer ormodify other physicochemical or functional properties on suchpolypeptides. According to certain embodiments, single or multiple aminoacid substitutions (in certain embodiments, conservative amino acidsubstitutions) may be made in the naturally-occurring sequence (incertain embodiments, in the portion of the polypeptide outside thedomain(s) forming intermolecular contacts).

In certain embodiments, a conservative amino acid substitution typicallymay not substantially change the structural characteristics of theparent sequence (e.g., a replacement amino acid should not tend to breaka helix that occurs in the parent sequence, or disrupt other types ofsecondary structure that characterizes the parent sequence). Examples ofart-recognized polypeptide secondary and tertiary structures aredescribed, e.g., in Proteins, Structures and Molecular Principles(Creighton, Ed., W. H. Freeman and Company, New York (1984));Introduction to Protein Structure (C. Branden and J. Tooze, eds.,Garland Publishing, New York, N.Y. (1991)); and Thornton et al. Nature354:105 (1991).

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion. In certainembodiments, fragments are at least 5 to 500 amino acids long. It willbe appreciated that in certain embodiments, fragments are at least 5, 6,8, 10, 14, 20, 50, 70, 100, 150, 200, 250, 300, 350, 400, 450, or 500amino acids long.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics.” Fauchere, J. Adv. Drug Res. 15:29(1986); Veber and Freidinger TINS p. 392 (1985); and Evans et al. J.Med. Chem. 30:1229 (1987). Such compounds are often developed with theaid of computerized molecular modeling. Peptide mimetics that arestructurally similar to therapeutically useful peptides may be used toproduce a similar therapeutic or prophylactic effect. Generally,peptidomimetics are structurally similar to a paradigm polypeptide(i.e., a polypeptide that has a biochemical property or pharmacologicalactivity), such as a human antibody, but have one or more peptidelinkages optionally replaced by a linkage selected from: —CH₂ NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH-(cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—, by methods well known in the art. Systematic substitution of one ormore amino acids of a consensus sequence with a D-amino acid of the sametype (e.g., D-lysine in place of L-lysine) may be used in certainembodiments to generate more stable peptides. In addition, constrainedpeptides comprising a consensus sequence or a substantially identicalconsensus sequence variation may be generated by methods known in theart (Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992)); for example,and not limitation, by adding internal cysteine residues capable offorming intramolecular disulfide bridges which cyclize the peptide.

The term “specific binding agent” refers to a natural or non-naturalmolecule that specifically binds to a target. Examples of specificbinding agents include, but are not limited to, proteins, peptides,nucleic acids, carbohydrates, lipids, and small molecule compounds. Incertain embodiments, a specific binding agent is an antibody. In certainembodiments, a specific binding agent is an antigen binding region.

The term “specifically binds” refers to the ability of a specificbinding agent to bind to a target with greater affinity than it binds toa non-target. In certain embodiments, specific binding refers to bindingto a target with an affinity that is at least 10, 50, 100, 250, 500, or1000 times greater than the affinity for a non-target. In certainembodiments, affinity is determined by an affinity ELISA assay. Incertain embodiments, affinity is determined by a BIAcore assay. Incertain embodiments, affinity is determined by a kinetic method. Incertain embodiments, affinity is determined by an equilibrium/solutionmethod.

The term “specific binding agent to TR-2” refers to a specific bindingagent that specifically binds any portion of TR-2. In certainembodiments, a specific binding agent to TR-2 is an antibody to TR-2. Incertain embodiments, a specific binding agent is an antigen bindingregion.

“Antibody” or “antibody peptide(s)” both refer to an intact antibody, ora fragment thereof. In certain embodiments, the antibody fragment may bea binding fragment that competes with the intact antibody for specificbinding. The term “antibody” also encompasses polyclonal antibodies andmonoclonal antibodies. In certain embodiments, binding fragments areproduced by recombinant DNA techniques. In certain embodiments, bindingfragments are produced by enzymatic or chemical cleavage of intactantibodies. In certain embodiments, binding fragments are produced byrecombinant DNA techniques. Binding fragments include, but are notlimited to, Fab, Fab′, F(ab′)2, Fv, and single-chain antibodies.Non-antigen binding fragments include, but are not limited to, Fcfragments. In certain embodiments, an antibody specifically binds to anepitope that is specifically bound by at least one antibody selectedfrom Ab A, Ab B, Ab C, Ab D, Ab E, Ab F, Ab G, Ab H, Ab I, Ab J, Ab K,Ab L, Ab M, Ab N, Ab O, Ab P, and Ab Q. The term “antibody” alsoencompasses anti-idiotypic antibodies that specifically bind to thevariable region of another antibody. In certain embodiments, ananti-idiotypic antibody specifically binds to the variable region of ananti-TR-2 antibody. In certain embodiments, anti-idiotypic antibodiesmay be used to detect the presence of a particular anti-TR-2 antibody ina sample or to block the activity of an anti-TR-2 antibody.

The term “anti-TR-2 antibody” as used herein means an antibody thatspecifically binds to TR-2. In certain embodiments, an anti-TR-2antibody binds to a TR-2 epitope to which at least one antibody selectedfrom Ab A to Q binds. In various embodiments, TR-2 may be the TR-2 ofany species, including, but not limited to, human, cynomolgus monkeys,mice, and rabbits. Certain assays for determining the specificity of anantibody are well known to the skilled artisan and include, but are notlimited to, ELISA, ELISPOT, western blots, BIAcore assays, solutionaffinity binding assays, T cell costimulation assays, and T cellmigration assays.

The term “isolated antibody” as used herein means an antibody which (1)is free of at least some proteins with which it would normally be found,(2) is essentially free of other proteins from the same source, e.g.,from the same species, (3) is expressed by a cell from a differentspecies, or (4) does not occur in nature.

The term “polyclonal antibody” refers to a heterogeneous mixture ofantibodies that bind to different epitopes of the same antigen.

The term “monoclonal antibodies” refers to a collection of antibodiesencoded by the same nucleic acid molecule. In certain embodiments,monoclonal antibodies are produced by a single hybridoma or other cellline, or by a transgenic mammal. Monoclonal antibodies typicallyrecognize the same epitope. The term “monoclonal” is not limited to anyparticular method for making an antibody.

The term “CDR grafted antibody” refers to an antibody in which the CDRfrom one antibody is inserted into the framework of another antibody. Incertain embodiments, the antibody from which the CDR is derived and theantibody from which the framework is derived are of different species.In certain embodiments, the antibody from which the CDR is derived andthe antibody from which the framework is derived are of differentisotypes.

The term “multi-specific antibody” refers to an antibody wherein two ormore variable regions bind to different epitopes. The epitopes may be onthe same or different targets. In certain embodiments, a multi-specificantibody is a “bi-specific antibody,” which recognizes two differentepitopes on the same or different antigens.

The term “catalytic antibody” refers to an antibody in which one or morecatalytic moieties is attached. In certain embodiments, a catalyticantibody is a cytotoxic antibody, which comprises a cytotoxic moiety.

The term “humanized antibody” refers to an antibody in which all or partof an antibody framework region is derived from a human, but all or partof one or more CDR regions is derived from another species, for examplea mouse.

The terms “human antibody” and “fully human antibody” are usedinterchangeably and refer to an antibody in which both the CDR and theframework comprise substantially human sequences. In certainembodiments, fully human antibodies are produced in non-human mammals,including, but not limited to, mice, rats, and lagomorphs. In certainembodiments, fully human antibodies are produced in hybridoma cells. Incertain embodiments, fully human antibodies are produced recombinantly.

In certain embodiments, an anti-TR-2 antibody comprises:

-   -   (i) a first polypeptide comprising at least one complementarity        determining region (CDR) selected from CDR1a, CDR2a, and CDR3a        -   wherein CDR1a comprises the amino acid sequence a b c d e f            g h i j k l, wherein amino acid a is glycine, amino acid b            is selected from glycine, tyrosine, or phenylalanine; amino            acid c is selected from serine or threonine; amino acid d is            selected from isoleucine or phenylalanine; amino acid e is            selected from serine, threonine, or asparagine; amino acid f            is selected from serine, aspartic acid, tyrosine,            asparagine, threonine, or glycine; amino acid g is selected            from glycine, aspartic acid, or tyrosine; amino acid h is            selected from glycine, aspartic acid, tyrosine, asparagine,            or serine; amino acid i is selected from tyrosine,            isoleucine, histidine, methionine, or tryptophan; amino acid            j is selected from asparagine, tyrosine, histidine, serine,            or phenylalanine; amino acid k is tryptophan or is not            present; and amino acid l is serine or is not present;        -   wherein CDR2a comprises the amino acid sequence m n o p q r            t u v w x y z a′ b′ c′, wherein amino acid m is selected            from tryptophan, tyrosine, histidine, valine, glutamic acid,            or serine; amino acid n is selected from methionine or            isoleucine; amino acid o is selected from asparagine,            tyrosine, serine, tryptophan, or histidine; amino acid p is            selected from proline, tyrosine, serine, arginine,            histidine, or asparagine; amino acid q is selected from            asparagine, serine, or aspartic acid; amino acid r is            selected from serine or glycine; amino acid s is selected            from aspartic acid, serine, threonine, or arginine; amino            acid t is selected from asparagine, threonine, alanine,            isoleucine, or tyrosine; amino acid u is selected from            threonine, tyrosine, leucine, lysine, asparagine, or            isoleucine; amino acid v is selected from glycine, tyrosine,            aspartic acid, or cysteine; amino acid w is selected from            tyrosine or asparagine; amino acid x is selected from            alanine or proline; amino acid y is selected from glutamine,            serine, or aspartic acid; amino acid z is selected from            lysine, leucine, or serine; amino acid a′ is selected from            phenylalanine, lysine, or valine; amino acid b′ is selected            from glutamine, serine, or lysine; and amino acid c′ is            glycine or is not present;        -   wherein CDR3a comprises the amino acid sequence d′ e′ f′ g′            h′ i′ j′ k′ l′ m′ n′ o′ p′ q′ r′ s′ t′ u′ v′ w′, wherein            amino acid d′ is selected from tryptophan, aspartic acid,            glycine, serine, or glutamic acid; amino acid e′ is selected            from asparagine, aspartic acid, glycine, arginine, serine,            valine, or leucine; amino acid f′ is selected from            histidine, serine, alanine, tyrosine, proline, asparagine,            glycine or threonine; amino acid g′ is selected from            tyrosine, serine, alanine, arginine, tryptophan, glycine or            valine; amino acid h′ is selected from glycine, alanine,            serine, asparagine, methionine, tyrosine, tryptophan,            cysteine, or aspartic acid; amino acid i′ is selected from            serine, tryptophan, glycine, phenylalanine, aspartic acid,            tyrosine, or threonine; amino acid j′ is selected from            glycine, threonine, serine, leucine, valine, asparagine,            tryptophan, or tyrosine; amino acid k′ is selected from            serine, phenylalanine, aspartic acid, tryptophan, glycine,            or tyrosine, or is not present; amino acid l′ is selected            from histidine, aspartic acid, alanine, tryptophan,            tyrosine, serine, phenylalanine, valine, or glycine, or is            not present; amino acid m′ is selected from phenylalanine,            tyrosine, glutamic acid, proline, aspartic acid, cysteine,            isoleucine, or methionine, or is not present; amino acid n′            is selected from aspartic acid, phenylalanine, alanine,            leucine, or serine, or is not present; amino acid o′ is            selected from tyrosine, leucine, aspartic acid,            phenylalanine, proline, or valine, or is not present; amino            acid p′ is selected from leucine, aspartic acid, or            tyrosine, or is not present; amino acid q′ is selected from            serine or tyrosine, or is not present; amino acid r′ is            tyrosine or is not present; amino acid s′ is selected from            glycine or tyrosine, or is not present; amino acid t′ is            selected from glycine or methionine, or is not present;            amino acid u′ is selected from methionine or aspartic acid,            or is not present; amino acid v′ is selected from aspartic            acid or valine, or is not present; and amino acid w′ is            valine or is not present; and        -   wherein the first polypeptide, in association with an            antibody light chain, binds TR-2; and    -   (ii) a second polypeptide comprising at least one        complementarity determining region (CDR) selected from CDR1b,        CDR2b, and CDR3b        -   wherein CDR1b comprises the amino acid sequence a1 b1 c1 d1            e1 f1 g1 h1 i1 j1 k1 l1 m1 n1 o1 p1 q1, wherein amino acid            a1 is selected from arginine or lysine; amino acid b1 is            selected from threonine, alanine, or serine; amino acid c1            is serine; amino acid d1 is glutamine; amino acid e1 is            selected from serine or glycine; amino acid f1 is selected            from isoleucine, leucine, or valine; amino acid g1 is            selected from serine, leucine, or arginine; amino acid h1 is            selected from threonine, serine, isoleucine, asparagine,            arginine, histidine, or tyrosine; amino acid i1 is selected            from tyrosine, arginine, tryptophan, aspartic acid, or            serine; j1 is selected from leucine, isoleucine, asparagine,            tyrosine, or serine; amino acid k1 is selected from            asparagine, glycine, valine, alanine, or leucine; amino acid            l1 is selected from tyrosine, alanine, or asparagine, or is            not present; amino acid m1 is selected from asparagine or            lysine, or is not present; amino acid n1 is selected from            tyrosine, asparagine, or isoleucine, or is not present;            amino acid o1 is selected from leucine or tyrosine, or is            not present; amino acid p1 is selected from aspartic acid or            leucine, or is not present; and amino acid q1 is selected            from valine, alanine, or threonine, or is not present;        -   wherein CDR2b comprises the amino acid sequence r1 s1 t1 u1            v1 w1 x1, wherein amino acid r1 is selected from alanine,            aspartic acid, leucine, tryptophan, glycine, or valine;            amino acid s1 is selected from threonine, valine, glycine,            or alanine; amino acid t1 is serine; amino acid u1 is            selected from serine, asparagine, or threonine; amino acid            v1 is selected from leucine, phenylalanine, or arginine;            amino acid w1 is selected from glutamine, alanine, or            glutamic acid; and amino acid x1 is selected from serine,            arginine, or threonine;        -   wherein CDR3b comprises the amino acid sequence y1 z1 a1′            b1′ c1′ d1′ e1′ f1′ g1′, wherein amino acid y1 is selected            from glutamine, methionine, leucine, or histidine; amino            acid z1 is selected from glutamine or lysine; amino acid a1′            is selected from serine, threonine, alanine, histidine,            tyrosine, or phenylalanine; amino acid b1′ is selected from            tyrosine, leucine, asparagine, or glycine; amino acid c1′ is            selected from serine, glutamine, isoleucine, or lysine;            amino acid d1′ is selected from threonine, phenylalanine,            tyrosine, alanine, or serine; amino acid e1′ is proline;            amino acid f1′ is selected from leucine, phenylalanine,            tryptophan, serine, or arginine; and amino acid g1′ is            selected from threonine or serine; and wherein the second            polypeptide, in association with an antibody heavy chain,            binds TR-2.

In certain embodiments, an anti-TR-2 antibody comprises: a firstpolypeptide comprising complementarity determining regions (CDRs) as setforth in SEQ ID NO: 2 and a second polypeptide comprising CDRs as setforth in SEQ ID NO: 36. In certain embodiments, an anti-TR-2 antibodycomprises: a first polypeptide comprising complementarity determiningregions (CDRs) as set forth in SEQ ID NO: 4 and a second polypeptidecomprising CDRs as set forth in SEQ ID NO: 38. In certain embodiments,an anti-TR-2 antibody comprises: a first polypeptide comprisingcomplementarity determining regions (CDRs) as set forth in SEQ ID NO: 6and a second polypeptide comprising CDRs as set forth in SEQ ID NO: 40.In certain embodiments, an anti-TR-2 antibody comprises: a firstpolypeptide comprising complementarity determining regions (CDRs) as setforth in SEQ ID NO: 8 and a second polypeptide comprising CDRs as setforth in SEQ ID NO: 42. In certain embodiments, an anti-TR-2 antibodycomprises: a first polypeptide comprising complementarity determiningregions (CDRs) as set forth in SEQ ID NO: 10 and a second polypeptidecomprising CDRs as set forth in SEQ ID NO: 44. In certain embodiments,an anti-TR-2 antibody comprises: a first polypeptide comprisingcomplementarity determining regions (CDRs) as set forth in SEQ ID NO: 12and a second polypeptide comprising CDRs as set forth in SEQ ID NO: 46.In certain embodiments, an anti-TR-2 antibody comprises: a firstpolypeptide comprising complementarity determining regions (CDRs) as setforth in SEQ ID NO: 14 and a second polypeptide comprising CDRs as setforth in SEQ ID NO: 48. In certain embodiments, an anti-TR-2 antibodycomprises: a first polypeptide comprising complementarity determiningregions (CDRs) as set forth in SEQ ID NO: 16 and a second polypeptidecomprising CDRs as set forth in SEQ ID NO: 50. In certain embodiments,an anti-TR-2 antibody comprises: a first polypeptide comprisingcomplementarity determining regions (CDRs) as set forth in SEQ ID NO: 18and a second polypeptide comprising CDRs as set forth in SEQ ID NO: 52.In certain embodiments, an anti-TR-2 antibody comprises: a firstpolypeptide comprising complementarity determining regions (CDRs) as setforth in SEQ ID NO: 20 and a second polypeptide comprising CDRs as setforth in SEQ ID NO: 54. In certain embodiments, an anti-TR-2 antibodycomprises: a first polypeptide comprising complementarity determiningregions (CDRs) as set forth in SEQ ID NO: 22 and a second polypeptidecomprising CDRs as set forth in SEQ ID NO: 56. In certain embodiments,an anti-TR-2 antibody comprises: a first polypeptide comprisingcomplementarity determining regions (CDRs) as set forth in SEQ ID NO: 24and a second polypeptide comprising CDRs as set forth in SEQ ID NO: 58.In certain embodiments, an anti-TR-2 antibody comprises: a firstpolypeptide comprising complementarity determining regions (CDRs) as setforth in SEQ ID NO: 26 and a second polypeptide comprising CDRs as setforth in SEQ ID NO: 60. In certain embodiments, an anti-TR-2 antibodycomprises: a first polypeptide comprising complementarity determiningregions (CDRs) as set forth in SEQ ID NO: 28 and a second polypeptidecomprising CDRs as set forth in SEQ ID NO: 62. In certain embodiments,an anti-TR-2 antibody comprises: a first polypeptide comprisingcomplementarity determining regions (CDRs) as set forth in SEQ ID NO: 30and a second polypeptide comprising CDRs as set forth in SEQ ID NO: 64.In certain embodiments, an anti-TR-2 antibody comprises: a firstpolypeptide comprising complementarity determining regions (CDRs) as setforth in SEQ ID NO: 32 and a second polypeptide comprising CDRs as setforth in SEQ ID NO: 66. In certain embodiments, an anti-TR-2 antibodycomprises: a first polypeptide comprising complementarity determiningregions (CDRs) as set forth in SEQ ID NO: 34 and a second polypeptidecomprising CDRs as set forth in SEQ ID NO: 68. In certain embodiments,an anti-TR-2 antibody comprises a first polypeptide as set forth inparagraph [079] above and a second polypeptide as set forth in paragraphabove. In certain embodiments, an anti-TR-2 antibody comprises a firstpolypeptide as set forth in paragraph [080] above and a secondpolypeptide as set forth in paragraph [085] above. In certainembodiments, an anti-TR-2 antibody is a human antibody. In certainembodiments, an anti-TR-2 antibody comprises a detectable label. Incertain embodiments, an anti-TR-2 antibody is a chimeric antibody.

“Chimeric antibody” refers to an antibody that has an antibody variableregion of a first species fused to another molecule, for example, anantibody constant region of another second species. See, e.g., U.S. Pat.No. 4,816,567 and Morrison et al., Proc Natl Acad Sci (USA),81:6851-6855 (1985). In certain embodiments, the first species may bedifferent from the second species. In certain embodiments, the firstspecies may be the same as the second species. In certain embodiments,chimeric antibodies may be made through mutagenesis or CDR grafting tomatch a portion of the known sequence of anti-TR-2 antibody variableregions. CDR grafting typically involves grafting the CDRs from anantibody with desired specificity onto the framework regions (FRs) ofanother antibody.

A bivalent antibody other than a “multispecific” or “multifunctional”antibody, in certain embodiments, typically is understood to have eachof its binding sites be identical.

An antibody substantially inhibits adhesion of a ligand to a receptorwhen an excess of antibody reduces the quantity of receptor bound to theligand by at least about 20%, 40%, 60%, 80%, 85%, or more (as measuredin an in vitro competitive binding assay).

The term “epitope” refers to a portion of a molecule capable of beingbound by a specific binding agent. Exemplary epitopes may comprise anypolypeptide determinant capable of specific binding to an immunoglobulinand/or T-cell receptor. Exemplary epitope determinants include, but arenot limited to, chemically active surface groupings of molecules, forexample, but not limited to, amino acids, sugar side chains, phosphorylgroups, and sulfonyl groups. In certain embodiments, epitopedeterminants may have specific three dimensional structuralcharacteristics, and/or specific charge characteristics. In certainembodiments, an epitope is a region of an antigen that is bound by anantibody. Epitopes may be contiguous or non-contiguous. In certainembodiments, epitopes may be mimetic in that they comprise a threedimensional structure that is similar to an epitope used to generate theantibody, yet comprise none or only some of the amino acid residuesfound in that epitope used to generate the antibody.

The term “inhibiting and/or neutralizing epitope” refers to an epitope,which when bound by a specific binding agent results in a decrease in abiological activity in vivo, in vitro, and/or in situ. In certainembodiments, a neutralizing epitope is located on or is associated witha biologically active region of a target.

The term “activating epitope” refers to an epitope, which when bound bya specific binding agent results in activation or maintenance of abiological activity in vivo, in vitro, and/or in situ. In certainembodiments, an activating epitope is located on or is associated with abiologically active region of a target.

In certain embodiments, an epitope is specifically bound by at least oneantibody selected from Ab A, Ab B, Ab C, Ab D, Ab E, Ab F, Ab G, Ab H,Ab I, Ab J, Ab K, Ab L, Ab M, Ab N, Ab O, Ab P, and Ab Q. In certainsuch embodiments, the epitope is substantially pure. In certain suchembodiments, the epitope is at a concentration of at least 1 nM. Incertain such embodiments, the epitope is at a concentration of between 1nM and 5 nM. In certain such embodiments, the epitope is at aconcentration of between 5 nM and 10 nM. In certain such embodiments,the epitope is at a concentration of between 10 nM and 15 nM.

In certain embodiments, an antibody specifically binds to an epitopethat is specifically bound by at least one antibody selected from Ab A,Ab B, Ab C, Ab D, Ab E, Ab F, Ab G, Ab H, Ab I, Ab J, Ab K, Ab L, Ab M,Ab N, Ab O, Ab P, and Ab Q, and is substantially pure. In certain suchembodiments, the antibody is at a concentration of at least 1 nM. Incertain such embodiments, the antibody is at a concentration of between1 nM and 5 nM. In certain such embodiments, the antibody is at aconcentration of between 5 nM and 10 nM. In certain such embodiments,the antibody is at a concentration of between 10 nM and 15 nM.

In certain embodiments, an antibody specifically binds to amino acids 1to 85 of mature human TR-2, and is substantially pure. In certain suchembodiments, the antibody is at a concentration of at least 1 nM. Incertain such embodiments, the antibody is at a concentration of between1 nM and 5 nM. In certain such embodiments, the antibody is at aconcentration of between 5 nM and 10 nM. In certain such embodiments,the antibody is at a concentration of between 10 nM and 15 nM.

In certain embodiments, an antibody competes for binding to an epitopewith at least one antibody selected from Ab A, Ab B, Ab C, Ab D, Ab E,Ab F, Ab G, Ab H, Ab I, Ab J, Ab K, Ab L, Ab M, Ab N, Ab O, Ab P, and AbQ. In certain such embodiments, the antibody is substantially pure. Incertain such embodiments, the antibody is at a concentration of at least1 nM. In certain such embodiments, the antibody is at a concentration ofbetween 1 nM and 5 nM. In certain such embodiments, the antibody is at aconcentration of between 5 nM and 10 nM. In certain such embodiments,the antibody is at a concentration of between 10 nM and 15 nM.

In certain embodiments, an antibody competes for binding to amino acids1 to 85 of mature human TR-2 with at least one antibody selected from AbA, Ab B, Ab C, Ab D, Ab E, Ab F, Ab G, Ab H, Ab I, Ab J, Ab K, Ab L, AbM, Ab N, Ab O, Ab P, and Ab Q. In certain such embodiments, the antibodyis substantially pure. In certain such embodiments, the antibody is at aconcentration of at least 1 nM. In certain such embodiments, theantibody is at a concentration of between 1 nM and 5 nM. In certain suchembodiments, the antibody is at a concentration of between 5 nM and 10nM. In certain such embodiments, the antibody is at a concentration ofbetween 10 nM and 15 nM.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

As used herein, the term “label” refers to any molecule that can bedetected. In a certain embodiment, an antibody may be labeled byincorporation of a radiolabeled amino acid. In a certain embodiment,biotin moieties that can be detected by marked avidin (e.g.,streptavidin containing a fluorescent marker or enzymatic activity thatcan be detected by optical or colorimetric methods) may be attached tothe antibody. In certain embodiments, a label may be incorporated intoor attached to another reagent which in turn binds to the antibody ofinterest. In certain embodiments, a label may be incorporated into orattached to an antibody that in turn specifically binds the antibody ofinterest. In certain embodiments, the label or marker can also betherapeutic. Various methods of labeling polypeptides and glycoproteinsare known in the art and may be used. Certain general classes of labelsinclude, but are not limited to, enzymatic, fluorescent,chemiluminescent, and radioactive labels. Certain examples of labels forpolypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., fluorescein isothiocyanate(FITC), rhodamine, lanthanide phosphors, phycoerythrin (PE)), enzymaticlabels (e.g., horseradish peroxidase, β-galactosidase, luciferase,alkaline phosphatase, glucose oxidase, glucose-6-phosphatedehydrogenase, alcohol dehydrogenase, malate dehydrogenase,penicillinase, luciferase), chemiluminescent labels, biotinyl groups,and predetermined polypeptide epitopes recognized by a secondaryreporter (e.g., leucine zipper pair sequences, binding sites forsecondary antibodies, metal binding domains, epitope tags). In certainembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance.

The term “sample”, as used herein, includes, but is not limited to, anyquantity of a substance from a living thing or formerly living thing.Such living things include, but are not limited to, humans, mice,monkeys, rats, rabbits, and other animals. Such substances include, butare not limited to, blood, serum, urine, cells, organs, tissues, bone,bone marrow, lymph nodes, and skin.

The term “pharmaceutical agent or drug” as used herein refers to achemical compound or composition capable of inducing a desiredtherapeutic effect when properly administered to a patient.

The term “modulator,” as used herein, is a compound that changes oralters the activity or function of a molecule. For example, a modulatormay cause an increase or decrease in the magnitude of a certain activityor function of a molecule compared to the magnitude of the activity orfunction observed in the absence of the modulator. In certainembodiments, a modulator is an inhibitor, which decreases the magnitudeof at least one activity or function of a molecule. Certain exemplaryactivities and functions of a molecule include, but are not limited to,binding affinity, enzymatic activity, and signal transduction. Certainexemplary inhibitors include, but are not limited to, proteins,peptides, antibodies, peptibodies, carbohydrates, and small organicmolecules. Exemplary peptibodies are described, e.g., in WO 01/83525.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition). In certainembodiments, a substantially purified fraction is a composition whereinthe object species comprises at least about 50 percent (on a molarbasis) of all macromolecular species present. In certain embodiments, asubstantially pure composition will comprise more than about 80%, 85%,90%, 95%, or 99% of all macromolar species present in the composition.In certain embodiments, the object species is purified to essentialhomogeneity (contaminant species cannot be detected in the compositionby conventional detection methods) wherein the composition consistsessentially of a single macromolecular species.

The term “patient” includes human and animal subjects.

According to certain embodiments, a cell line expressing anti-TR-2antibodies is provided.

In certain embodiments, chimeric antibodies that comprise at least aportion of a human sequence and another species' sequence are provided.In certain embodiments, such a chimeric antibody may result in a reducedimmune response in a host than an antibody without that host's antibodysequences. For example, in certain instances, an animal of interest maybe used as a model for a particular human disease. To study the effectof an antibody on that disease in the animal host, one could use anantibody from a different species. But, in certain instances, suchantibodies from another species, may elicit an immune response to theantibodies themselves in the host animal, thus impeding evaluation ofthese antibodies. In certain embodiments, replacing part of the aminoacid sequence of an anti-TR-2 antibody with antibody amino acid sequencefrom the host animal may decrease the magnitude of the host animal'santi-antibody response.

In certain embodiments, a chimeric antibody comprises a heavy chain anda light chain, wherein the variable regions of the light chain and theheavy chain are from a first species and the constant regions of thelight chain and the heavy chain are from a second species. In certainembodiments, the antibody heavy chain constant region is an antibodyheavy chain constant region of a species other than human. In certainembodiments, the antibody light chain constant region is an antibodylight chain constant region of a species other than human. In certainembodiments, the antibody heavy chain constant region is a humanantibody heavy chain constant region, and the antibody heavy chainvariable region is an antibody heavy chain variable region of a speciesother than human. In certain embodiments, the antibody light chainconstant region is a human antibody light chain constant region, and theantibody light chain variable region is an antibody light chain variableregion of a species other than human. Exemplary antibody constantregions include, but are not limited to, a human antibody constantregion, a cynomolgus monkey antibody constant region, a mouse antibodyconstant region, and a rabbit antibody constant region. Exemplaryantibody variable regions include, but are not limited to, a humanantibody variable region, a mouse antibody variable region, a pigantibody variable region, a guinea pig antibody variable region, acynomolgus monkey antibody variable region, and a rabbit antibodyvariable region. In certain embodiments, the framework regions of thevariable region in the heavy chain and light chain may be replaced withframework regions derived from other antibody sequences.

Certain exemplary chimeric antibodies may be produced by methods wellknown to those of ordinary skill in the art. In certain embodiments, thepolynucleotide of the first species encoding the heavy chain variableregion and the polynucleotide of the second species encoding the heavychain constant region can be fused. In certain embodiments, thepolynucleotide of the first species encoding the light chain variableregion and the nucleotide sequence of the second species encoding thelight chain constant region can be fused. In certain embodiments, thesefused nucleotide sequences can be introduced into a cell either in asingle expression vector (e.g., a plasmid) or in multiple expressionvectors. In certain embodiments, a cell comprising at least oneexpression vector may be used to make polypeptide. In certainembodiments, these fused nucleotide sequences can be introduced into acell either in separate expression vectors or in a single expressionvector. In certain embodiments, the host cell expresses both the heavychain and the light chain, which combine to produce an antibody. Incertain embodiments, a cell comprising at least one expression vectormay be used to make an antibody. Exemplary methods for producing andexpressing antibodies are discussed below.

In certain embodiments, conservative modifications to the heavy andlight chains of an anti-TR-2 antibody (and corresponding modificationsto the encoding nucleotides) will produce antibodies having functionaland chemical characteristics similar to those of the original antibody.In contrast, in certain embodiments, substantial modifications in thefunctional and/or chemical characteristics of an anti-TR-2 antibody maybe accomplished by selecting substitutions in the amino acid sequence ofthe heavy and light chains that differ significantly in their effect onmaintaining (a) the structure of the molecular backbone in the area ofthe substitution, for example, as a sheet or helical conformation, (b)the charge or hydrophobicity of the molecule at the target site, or (c)the bulk of the side chain.

Certain desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. In certain embodiments, amino acidsubstitutions can be used to identify important residues of theanti-TR-2 antibodies, such as those which may increase or decrease theaffinity of the antibodies to TR-2 or the effector function of theantibodies.

In certain embodiments, the effects of an anti-TR-2 antibody may beevaluated by measuring a reduction in the amount of symptoms of thedisease. In certain embodiments, the disease of interest may be causedby a pathogen. In certain embodiments, a disease may be established inan animal host by other methods including introduction of a substance(such as a carcinogen) and genetic manipulation. In certain embodiments,effects may be evaluated by detecting one or more adverse events in theanimal host. The term “adverse event” includes, but is not limited to,an adverse reaction in an animal host that receives an antibody that isnot present in an animal host that does not receive the antibody. Incertain embodiments, adverse events include, but are not limited to, afever, an immune response to an antibody, inflammation, and/or death ofthe animal host.

Various antibodies specific to an antigen may be produced in a number ofways. In certain embodiments, an antigen containing an epitope ofinterest may be introduced into an animal host (e.g., a mouse), thusproducing antibodies specific to that epitope. In certain instances,antibodies specific to an epitope of interest may be obtained frombiological samples taken from hosts that were naturally exposed to theepitope. In certain instances, introduction of human immunoglobulin (Ig)loci into mice in which the endogenous Ig genes have been inactivatedoffers the opportunity to obtain human monoclonal antibodies (MAbs).

Naturally Occurring Antibody Structure

Naturally occurring antibody structural units typically comprise atetramer. Each such tetramer typically is composed of two identicalpairs of polypeptide chains, each pair having one full-length “light”chain (in certain embodiments, about 25 kDa) and one full-length “heavy”chain (in certain embodiments, about 50-70 kDa). The term “heavy chain”includes any polypeptide having sufficient variable region sequence toconfer specificity for a particular antigen. A full-length heavy chainincludes a variable region domain, V_(H), and three constant regiondomains, C_(H)1, C_(H)2, and C_(H)3. The V_(H) domain is at theamino-terminus of the polypeptide, and the C_(H)3 domain is at thecarboxy-terminus. The term “heavy chain”, as used herein, encompasses afull-length antibody heavy chain and fragments thereof.

The term “light chain” includes any polypeptide having sufficientvariable region sequence to confer specificity for a particular antigen.A full-length light chain includes a variable region domain, V_(L), anda constant region domain, C_(L). Like the heavy chain, the variableregion domain of the light chain is at the amino-terminus of thepolypeptide. The term “light chain”, as used herein, encompasses afull-length light chain and fragments thereof.

The amino-terminal portion of each chain typically includes a variableregion (V_(H) in the heavy chain and V_(L) in the light chain) of about100 to 110 or more amino acids that typically is responsible for antigenrecognition. The carboxy-terminal portion of each chain typicallydefines a constant region (C_(H) domains in the heavy chain and C_(L) inthe light chain) that may be responsible for effector function. Antibodyeffector functions include activation of complement and stimulation ofopsonophagocytosis. Human light chains are typically classified as kappaand lambda light chains. Heavy chains are typically classified as mu,delta, gamma, alpha, or epsilon, and define the antibody's isotype asIgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses,including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM hassubclasses including, but not limited to, IgM1 and IgM2. IgA issimilarly subdivided into subclasses including, but not limited to, IgA1and IgA2. Within full-length light and heavy chains, typically, thevariable and constant regions are joined by a “J” region of about 12 ormore amino acids, with the heavy chain also including a “D” region ofabout 10 more amino acids. See, e.g., Fundamental Immunology Ch. 7(Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). The variable regionsof each light/heavy chain pair typically form the antigen binding site.

The variable regions typically exhibit the same general structure ofrelatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor CDRs. The CDRs from the heavy and light chains of each pair typicallyare aligned by the framework regions, which may enable binding to aspecific epitope. From N-terminal to C-terminal, both light and heavychain variable regions typically comprise the domains FR1, CDR1, FR2,CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domainis typically in accordance with the definitions of Kabat Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol.196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989).

As discussed above, there are several types of antibody fragments. A Fabfragment is comprised of one light chain and the C_(H)1 and variableregions of one heavy chain. The heavy chain of a Fab molecule cannotform a disulfide bond with another heavy chain molecule. A Fab′ fragmentcontains one light chain and one heavy chain that contains more of theconstant region, between the C_(H)1 and C_(H)2 domains, such that aninterchain disulfide bond can be formed between two heavy chains to forma F(ab′)2 molecule. A Fab fragment is similar to a F(ab′)2 molecule,except the constant region in the heavy chains of the molecule extendsto the end of the C_(H)2 domain. The Fv region comprises the variableregions from both the heavy and light chains, but lacks the constantregions. Single-chain antibodies are Fv molecules in which the heavy andlight chain variable regions have been connected by a flexible linker toform a single polypeptide chain which forms an antigen-binding region.Exemplary single chain antibodies are discussed in detail, e.g., in WO88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203. A Fc fragmentcontains the C_(H)2 and C_(H)3 domains of the heavy chain and containsmore of the constant region, between the C_(H)1 and C_(H)2 domains, suchthat an interchain disulfide bond can be formed between two heavychains.

In certain embodiments, functional domains, C_(H)1, C_(H)2, C_(H)3, andintervening sequences can be shuffled to create a different antibodyconstant region. For example, in certain embodiments, such hybridconstant regions can be optimized for half-life in serum, for assemblyand folding of the antibody tetramer, and/or for improved effectorfunction. In certain embodiments, modified antibody constant regions maybe produced by introducing single point mutations into the amino acidsequence of the constant region and testing the resulting antibody forimproved qualities, e.g., one or more of those listed above.

In certain embodiments, an antibody of one isotype is converted to adifferent isotype by isotype switching without losing its specificityfor a particular target molecule. Methods of isotype switching include,but are not limited to, direct recombinant techniques (see e.g., U.S.Pat. No. 4,816,397) and cell-cell fusion techniques (see e.g., U.S. Pat.No. 5,916,771), among others. In certain embodiments, an antibody can beconverted from one subclass to another subclass using techniquesdescribed above or otherwise known in the art without losing itsspecificity for a particular target molecule, including, but not limitedto, conversion from an IgG2 subclass to an IgG1, IgG3, or IgG4 subclass.

Bispecific or Bifunctional Antibodies

A bispecific or bifunctional antibody typically is an artificial hybridantibody having two different heavy/light chain pairs and two differentbinding sites. Bispecific antibodies may be produced by a variety ofmethods including, but not limited to, fusion of hybridomas or linkingof Fab′ fragments. See, e.g., Songsivilai & Lachmann Clin. Exp. Immunol.79: 315-321 (1990), Kostelny et al. J. Immunol. 148:1547-1553 (1992).

Certain Preparation of Antibodies

In certain embodiments, antibodies can be expressed in cell lines otherthan hybridoma cell lines. In certain embodiments, sequences encodingparticular antibodies, including chimeric antibodies, can be used fortransformation of a suitable mammalian host cell. According to certainembodiments, transformation can be by any known method for introducingpolynucleotides into a host cell, including, for example packaging thepolynucleotide in a virus (or into a viral vector) and transducing ahost cell with the virus or by transfecting a vector using proceduresknown in the art, as exemplified by U.S. Pat. Nos. 4,399,216; 4,912,040;4,740,461; and 4,959,455.

In certain embodiments, an expression vector comprises any of thepolynucleotide sequences discussed herein. In certain embodiments, amethod of making a polypeptide comprising producing the polypeptide in acell comprising any of the above expression vectors in conditionssuitable to express the polynucleotide contained therein to produce thepolypeptide is provided.

In certain embodiments, an expression vector comprises a polynucleotidecomprising a sequence encoding a polypeptide comprising at least onecomplementarity determining region (CDR) selected from CDR1a, CDR2a, andCDR3a, wherein CDR1a comprises the amino acid sequence a b c d e f g h ij k l, wherein amino acid a is glycine, amino acid b is selected fromglycine, tyrosine, or phenylalanine; amino acid c is selected fromserine or threonine; amino acid d is selected from isoleucine orphenylalanine; amino acid e is selected from serine, threonine, orasparagine; amino acid f is selected from serine, aspartic acid,tyrosine, asparagine, threonine, or glycine; amino acid g is selectedfrom glycine, aspartic acid, or tyrosine; amino acid h is selected fromglycine, aspartic acid, tyrosine, asparagine, or serine; amino acid i isselected from tyrosine, isoleucine, histidine, methionine, ortryptophan; amino acid j is selected from asparagine, tyrosine,histidine, serine, or phenylalanine; amino acid k is tryptophan or isnot present; and amino acid l is serine or is not present; wherein CDR2acomprises the amino acid sequence m n o p q r s t u v w x y z a′ b′ c′,wherein amino acid m is selected from tryptophan, tyrosine, histidine,valine, glutamic acid, or serine; amino acid n is selected frommethionine or isoleucine; amino acid o is selected from asparagine,tyrosine, serine, tryptophan, or histidine; amino acid p is selectedfrom proline, tyrosine, serine, arginine, histidine, or asparagine;amino acid q is selected from asparagine, serine, or aspartic acid;amino acid r is selected from serine or glycine; amino acid s isselected from aspartic acid, serine, threonine, or arginine; amino acidt is selected from asparagine, threonine, alanine, isoleucine, ortyrosine; amino acid u is selected from threonine, tyrosine, leucine,lysine, asparagine, or isoleucine; amino acid v is selected fromglycine, tyrosine, aspartic acid, or cysteine; amino acid w is selectedfrom tyrosine or asparagine; amino acid x is selected from alanine orproline; amino acid y is selected from glutamine, serine, or asparticacid; amino acid z is selected from lysine, leucine, or serine; aminoacid a′ is selected from phenylalanine, lysine, or valine; amino acid b′is selected from glutamine, serine, or lysine; and amino acid c′ isglycine or is not present; wherein CDR3a comprises the amino acidsequence d′ e′ f′ g′ h′ i′ j′ k′ l′ m′ n′ o′ p′ q′ r′ s′ t′ u, v′ w′,wherein amino acid d′ is selected from tryptophan, aspartic acid,glycine, serine, or glutamic acid; amino acid e′ is selected fromasparagine, aspartic acid, glycine, arginine, serine, valine, orleucine; amino acid f′ is selected from histidine, serine, alanine,tyrosine, proline, asparagine, glycine or threonine; amino acid g′ isselected from tyrosine, serine, alanine, arginine, tryptophan, glycineor valine; amino acid h′ is selected from glycine, alanine, serine,asparagine, methionine, tyrosine, tryptophan, cysteine, or asparticacid; amino acid i′ is selected from serine, tryptophan, glycine,phenylalanine, aspartic acid, tyrosine, or threonine; amino acid j′ isselected from glycine, threonine, serine, leucine, valine, asparagine,tryptophan, or tyrosine; amino acid k′ is selected from serine,phenylalanine, aspartic acid, tryptophan, glycine, or tyrosine, or isnot present; amino acid l′ is selected from histidine, aspartic acid,alanine, tryptophan, tyrosine, serine, phenylalanine, valine, orglycine, or is not present; amino acid m′ is selected fromphenylalanine, tyrosine, glutamic acid, proline, aspartic acid,cysteine, isoleucine, or methionine, or is not present; amino acid n′ isselected from aspartic acid, phenylalanine, alanine, leucine, or serine,or is not present; amino acid o′ is selected from tyrosine, leucine,aspartic acid, phenylalanine, proline, or valine, or is not present;amino acid p′ is selected from leucine, aspartic acid, or tyrosine, oris not present; amino acid q′ is selected from serine or tyrosine, or isnot present; amino acid r′ is tyrosine or is not present; amino acid s′is selected from glycine or tyrosine, or is not present; amino acid t′is selected from glycine or methionine, or is not present; amino acid u′is selected from methionine or aspartic acid, or is not present; aminoacid v′ is selected from aspartic acid or valine, or is not present; andamino acid w′ is valine or is not present; and wherein the polypeptide,in association with an antibody light chain, binds TR-2. In certainembodiments, a method of making a polypeptide comprising producing thepolypeptide in a cell comprising the above expression vector inconditions suitable to express the polynucleotide contained therein toproduce the polypeptide is provided.

In certain embodiments, an expression vector comprises a polynucleotidecomprising a sequence encoding a polypeptide comprising at least onecomplementarity determining region (CDR) selected from CDR1b, CDR2b, andCDR3b, wherein CDR1b comprises the amino acid sequence a1 b1 c1 d1 e1 f1g1 h1 i1 j1 k1 l1 m1 n1 o1 p1 q1, wherein amino acid a1 is selected fromarginine or lysine; amino acid b1 is selected from threonine, alanine,or serine; amino acid c1 is serine; amino acid d1 is glutamine; aminoacid e1 is selected from serine or glycine; amino acid f1 is selectedfrom isoleucine, leucine, or valine; amino acid g1 is selected fromserine, leucine, or arginine; amino acid h1 is selected from threonine,serine, isoleucine, asparagine, arginine, histidine, or tyrosine; aminoacid i1 is selected from tyrosine, arginine, tryptophan, aspartic acid,or serine; j1 is selected from leucine, isoleucine, asparagine,tyrosine, or serine; amino acid k1 is selected from asparagine, glycine,valine, alanine, or leucine; amino acid l1 is selected from tyrosine,alanine, or asparagine, or is not present; amino acid m1 is selectedfrom asparagine or lysine, or is not present; amino acid n1 is selectedfrom tyrosine, asparagine, or isoleucine, or is not present; amino acido1 is selected from leucine or tyrosine, or is not present; amino acidp1 is selected from aspartic acid or leucine, or is not present; andamino acid q1 is selected from valine, alanine, or threonine, or is notpresent; wherein CDR2b comprises the amino acid sequence r1 s1 t1 u1 v1w1 x1, wherein amino acid r1 is selected from alanine, aspartic acid,leucine, tryptophan, glycine, or valine; amino acid s1 is selected fromthreonine, valine, glycine, or alanine; amino acid t1 is serine; aminoacid u1 is selected from serine, asparagine, or threonine; amino acid v1is selected from leucine, phenylalanine, or arginine; amino acid w1 isselected from glutamine, alanine, or glutamic acid; and amino acid x1 isselected from serine, arginine, or threonine; wherein CDR3b comprisesthe amino acid sequence y1 z1 a1′ b1′ c1′ d1′ e1′ f1′ g1′, wherein aminoacid y1 is selected from glutamine, methionine, leucine, or histidine;amino acid z1 is selected from glutamine or lysine; amino acid a1′ isselected from serine, threonine, alanine, histidine, tyrosine, orphenylalanine; amino acid b1′ is selected from tyrosine, leucine,asparagine, or glycine; amino acid c1′ is selected from serine,glutamine, isoleucine, or lysine; amino acid d1′ is selected fromthreonine, phenylalanine, tyrosine, alanine, or serine; amino acid e1′is proline; amino acid f1′ is selected from leucine, phenylalanine,tryptophan, serine, or arginine; and amino acid g1′ is selected fromthreonine or serine; and wherein the polypeptide, in association with anantibody heavy chain, binds TR-2. In certain embodiments, a method ofmaking a polypeptide comprising producing the polypeptide in a cellcomprising the above expression vector in conditions suitable to expressthe polynucleotide contained therein to produce the polypeptide isprovided. In certain embodiments, a cell comprising at least one of theabove expression vectors is provided. In certain embodiments, a methodof making an polypeptide comprising producing the polypeptide in a cellcomprising the above expression vector in conditions suitable to expressthe polynucleotide contained therein to produce the polypeptide isprovided.

In certain embodiments, an expression vector expresses an anti-TR-2antibody heavy chain. In certain embodiments, an expression vectorexpresses an anti-TR-2 antibody light chain. In certain embodiments, anexpression vector expresses both an anti-TR-2 antibody heavy chain andan anti-TR-2 antibody light chain. In certain embodiments, a method ofmaking an anti-TR-2 antibody comprising producing the antibody in a cellcomprising at least one of the expression vectors described herein inconditions suitable to express the polynucleotides contained therein toproduce the antibody is provided.

In certain embodiments, the transfection procedure used may depend uponthe host to be transformed. Certain methods for introduction ofheterologous polynucleotides into mammalian cells are known in the artand include, but are not limited to, dextran-mediated transfection,calcium phosphate precipitation, polybrene mediated transfection,protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

Certain mammalian cell lines available as hosts for expression are knownin the art and include, but are not limited to, many immortalized celllines available from the American Type Culture Collection (ATCC),including but not limited to Chinese hamster ovary (CHO) cells, E5cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells(COS), human hepatocellular carcinoma cells (e.g., Hep G2), NS0 cells,SP20 cells, Per C6 cells, 293 cells, and a number of other cell lines.In certain embodiments, cell lines may be selected through determiningwhich cell lines have high expression levels and produce antibodies withconstitutive antigen binding properties.

In certain embodiments, the vectors that may be transfected into a hostcell comprise control sequences that are operably linked to apolynucleotide encoding an anti-TR-2 antibody. In certain embodiments,control sequences facilitate expression of the linked polynucleotide,thus resulting in the production of the polypeptide encoded by thelinked polynucleotide. In certain embodiments, the vector also comprisespolynucleotide sequences that allow chromosome-independent replicationin the host cell. Exemplary vectors include, but are not limited to,plasmids (e.g., BlueScript, puc, etc.), cosmids, and YACS.

Certain Antibody Uses

According to certain embodiments, antibodies are useful for detecting aparticular antigen in a sample. In certain embodiments, this allows theidentification of cells or tissues which produce the protein. Forexample, in certain embodiments, anti-TR-2 antibodies may be used todetect the presence of TR-2 in a sample. In certain embodiments, amethod for detecting the presence or absence of TR-2 in a samplecomprises (a) combining an anti-TR-2 antibody and the sample; (b)separating antibodies bound to an antigen from unbound antibodies; and(c) detecting the presence or absence of antibodies bound to theantigen.

Assays in which an antibody may be used to detect the presence orabsence of an antigen include, but are not limited to, an ELISA and awestern blot. In certain embodiments, an anti-TR-2 antibody may belabeled. In certain embodiments, an anti-TR-2 antibody may be detectedby a labeled antibody that binds to the anti-TR-2 antibody. In certainembodiments, a kit for detecting the presence or absence of TR-2 in asample is provided. In certain embodiments, the kit comprises ananti-TR-2 antibody and reagents for detecting the antibody.

In certain embodiments, antibodies may be used to substantially isolatea chemical moiety such as, but not limited to, a protein. In certainembodiments, the antibody is attached to a “substrate,” which is asupporting material used for immobilizing the antibody. Substratesinclude, but are not limited to, tubes, plates (i.e., multi-wellplates), beads such as microbeads, filters, balls, and membranes. Incertain embodiments, a substrate can be made of water-insolublematerials such as, but not limited to, polycarbonate resin, siliconeresin, or nylon resin. Exemplary substrates for use in affinitychromatography include, but are not limited to, cellulose, agarose,polyacrylamide, dextran, polystyrene, polyvinyl alcohol, and poroussilica. There are many commercially available chromatography substratesthat include, but are not limited to, Sepharose 2B, Sepharose 4B,Sepharose 6B and other forms of Sepharose (Pharmacia); Bio-Gel (andvarious forms of Bio-Gel such as Biogel A, P, or CM), Cellex (andvarious forms of Cellex such as Cellex AE or Cellex-CM), Chromagel A,Chromagel P and Enzafix (Wako Chemical Indus.). The use of antibodyaffinity columns is known to a person of ordinary skill in the art. Incertain embodiments, a method for isolating TR-2 comprises (a) attachinga TR-2 antibody to a substrate; (b) exposing a sample containing TR-2 tothe antibody of part (a); and (c) isolating TR-2. In certainembodiments, a kit for isolating TR-2 is provided. In certainembodiments, the kit comprises an anti-TR-2 antibody attached to asubstrate and reagents for isolating TR-2.

The term “affinity chromatography” as used herein means a method ofseparating or purifying the materials of interest in a sample byutilizing the interaction (e.g., the affinity) between a pair ofmaterials, such as an antigen and an antibody, an enzyme and asubstrate, or a receptor and a ligand.

In certain embodiments, antibodies which bind to a particular proteinand block interaction with other binding compounds may have therapeuticuse. In this application, when discussing the use of anti-TR-2antibodies to treat diseases or conditions, such use may include use ofthe anti-TR-2 antibodies themselves; compositions comprising anti-TR-2antibodies; and/or combination therapies comprising anti-TR-2 antibodiesand one or more additional active ingredients. When anti-TR-2 antibodiesare used to “treat” a disease or condition, such treatment may or maynot include prevention of the disease or condition. In certainembodiments, anti-TR-2 antibodies can block the interaction of the TR-2receptor with its ligand, TRAIL. In certain embodiments, anti-TR-2antibodies can activate the TR-2 receptor. In certain embodiments,anti-TR-2 antibodies can constitutively activate the TR-2 receptor.Because TR-2 is associated with apoptosis, in certain embodiments,anti-TR-2 antibodies may have therapeutic use in treating diseases inwhich cell death or prevention of cell death is desired. Such diseasesinclude, but are not limited to, cancer associated with any tissueexpressing TR-2, inflammation, and viral infections.

In certain embodiments, an anti-TR-2 antibody is administered alone. Incertain embodiments, an anti-TR-2 antibody is administered prior to theadministration of at least one other therapeutic agent. In certainembodiments, an anti-TR-2 antibody is administered concurrent with theadministration of at least one other therapeutic agent. In certainembodiments, an anti-TR-2 antibody is administered subsequent to theadministration of at least one other therapeutic agent. Exemplarytherapeutic agents, include, but are not limited to, at least one othercancer therapy agent. Exemplary cancer therapy agents include, but arenot limited to, radiation therapy and chemotherapy.

In certain embodiments, anti-TR-2 antibody pharmaceutical compositionscan be administered in combination therapy, i.e., combined with otheragents. In certain embodiments, the combination therapy comprises ananti-TR-2 antibody, in combination with at least one anti-angiogenicagent. Exemplary agents include, but are not limited to, in vitrosynthetically prepared chemical compositions, antibodies, antigenbinding regions, radionuclides, and combinations and conjugates thereof.In certain embodiments, an agent may act as an agonist, antagonist,allosteric modulator, or toxin. In certain embodiments, an agent may actto inhibit or stimulate its target (e.g., receptor or enzyme activationor inhibition), and thereby promote cell death or arrest cell growth.

Exemplary chemotherapy treatments include, but are not limited toanti-neoplastic agents including, but not limited to, alkylating agentsincluding, but not limited to: nitrogen mustards, including, but notlimited to, mechlorethamine, cyclophosphamide, ifosfamide, melphalan andchlorambucil; nitrosoureas, including, but not limited to, carmustineBCNU, lomustine, CCNU, and semustine, methyl-CCNU; Temodal™,temozolomide; ethylenimines/methylmelamine, including, but not limitedto, triethylenemelamine (TEM), triethylene, thiophosphoramide, thiotepa,hexamethylmelamine (HMM), and altretamine; alkyl sulfonates, including,but not limited to, busulfan; triazines, including, but not limited to,dacarbazine (DTIC); antimetabolites, including, but not limited to,folic acid analogs such as methotrexate and trimetrexate; pyrimidineanalogs, including, but not limited to, 5-fluorouracil (5FU),fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC,cytarabine), 5-azacytidine, and 2,2′-difluorodeoxycytidine; purineanalogs, including, but not limited to, 6-mercaptopurine, 6-thioguanine,azathioprine, 2′-deoxycoformycin (pentostatin),erythrohydroxynonyladenine (EHNA), fludarabine phosphate, cladribine,and 2-chlorodeoxyadenosine (2-CdA); natural products, including, but notlimited to, antimitotic drugs such as paclitaxel; vinca alkaloids,including, but not limited to, vinblastine (VLB), vincristine, andvinorelbine; taxotere; estramustine and estramustine phosphate;pipodophylotoxins, including, but not limited to, etoposide andteniposide; antibiotics, including, but not limited to, actinomycin D,daunomycin, rubidomycin, doxorubicin, mitoxantrone, idarubicin,bleomycins, plicamycin, mithramycin, mitomycin C, and actinomycin;enzymes, including, but not limited to, L-asparaginase; biologicalresponse modifiers, including, but not limited to, interferon-alpha,IL-2, G-CSF, and GM-CSF; doxycycline; irinotecan hydrochloride;miscellaneous agents, including, but not limited to, platinumcoordination complexes such as cisplatin and carboplatin;anthracenediones, including, but not limited to, mitoxantrone;substituted urea, including, but not limited to, hydroxyurea;methylhydrazine derivatives, including, but not limited to,N-methylhydrazine (MIH) and procarbazine; adrenocortical suppressants,including, but not limited to, mitotane (o,p′-DDD) andaminoglutethimide; hormones and antagonists, including, but not limitedto, adrenocorticosteroid antagonists such as prednisone and equivalents,dexamethasone and aminoglutethimide; Gemzar™, gemcitabine; progestin,including, but not limited to, hydroxyprogesterone caproate,medroxyprogesterone acetate and megestrol acetate; estrogen, including,but not limited to, diethylstilbestrol and ethinyl estradiolequivalents; antiestrogen, including, but not limited to, tamoxifen;androgens, including, but not limited to, testosterone propionate andfluoxymesterone/equivalents; antiandrogens, including, but not limitedto, flutamide, gonadotropin-releasing hormone analogs and leuprolide;and non-steroidal antiandrogens, including, but not limited to,flutamide.

Exemplary cancer therapies, which may be administered with an anti-TR-2antibody, include, but are not limited to, targeted therapies. Examplesof targeted therapies include, but are not limited to, use oftherapeutic antibodies. Exemplary therapeutic antibodies, include, butare not limited to, mouse, mouse-human chimeric, CDR-grafted, humanized,and human antibodies, and synthetic antibodies, including, but notlimited to, those selected by screening antibody libraries. Exemplaryantibodies include, but are not limited to, those which bind to cellsurface proteins Her2, CDC20, CDC33, mucin-like glycoprotein, andepidermal growth factor receptor (EGFr) present on tumor cells, andoptionally induce a cytostatic and/or cytotoxic effect on tumor cellsdisplaying these proteins. Exemplary antibodies also include, but arenot limited to, HERCEPTIN™, trastuzumab, which may be used to treatbreast cancer and other forms of cancer; RITUXAN™, rituximab, ZEVALIN™,ibritumomab tiuxetan, and LYMPHOCIDE™, epratuzumab, which may be used totreat non-Hodgkin's lymphoma and other forms of cancer; GLEEVEC™,imatinib mesylate, which may be used to treat chronic myeloid leukemiaand gastrointestinal stromal tumors; and BEXXAR™, iodine 131tositumomab, which may be used for treatment of non-Hodgkin's lymphoma.Certain exemplary antibodies also include ERBITUX™; IMC-C225; Iressa™;gefitinib; TARCEVA™, ertinolib; KDR (kinase domain receptor) inhibitors;anti VEGF antibodies and antagonists (e.g., Avastin™ and VEGAF-TRAP);anti VEGF receptor antibodies and antigen binding regions; anti-Ang-1and Ang-2 antibodies and antigen binding regions; antibodies to Tie-2and other Ang-1 and Ang-2 receptors; Tie-2 ligands; antibodies againstTie-2 kinase inhibitors; and Campath®, alemtuzumab. In certainembodiments, cancer therapy agents are other polypeptides whichselectively induce apoptosis in tumor cells, including, but not limitedto, TNF-related polypeptides such as TRAIL.

In certain embodiments, specific binding agents (including, but notlimited to, anti-IGF-R1 antibodies) that antagonize the binding of theligands IGF-1 and/or IGF-2 to insulin-like growth factor-1 receptor(“IGF-1R”) and promote apoptosis of cells expressing IGF-1R areformulated or administered in combination with specific binding agents(including, but not limited to, TRAIL and anti-TR2 antibodies) thatagonize and thereby promote apoptosis of cells expressing TRAIL-R2.Exemplary anti-IGF-1R antibodies are known in the art and are disclosed,for example, in WO 2006/069202, filed Dec. 20, 2005, which isincorporated by reference herein for any purpose.

In certain embodiments, cancer therapy agents are anti-angiogenic agentswhich decrease angiogenesis. Certain such agents include, but are notlimited to, ERBITUX™, IMC-C225; KDR (kinase domain receptor) inhibitoryagents (e.g., antibodies and antigen binding regions that specificallybind to the kinase domain receptor); anti-VEGF agents (e.g., antibodiesor antigen binding regions that specifically bind VEGF, or soluble VEGFreceptors or a ligand binding region thereof) such as AVASTIN™ orVEGF-TRAP™; anti-VEGF receptor agents (e.g., antibodies or antigenbinding regions that specifically bind thereto); EGFR inhibitory agents(e.g., antibodies or antigen binding regions that specifically bindthereto) such as ABX-EGF, panitumumab, IRESSA™, gefitinib, TARCEVA™,erlotinib, anti-Ang1 and anti-Ang2 agents (e.g., antibodies or antigenbinding regions specifically binding thereto or to their receptors,e.g., Tie2/Tek); and anti-Tie-2 kinase inhibitory agents (e.g.,antibodies or antigen binding regions that specifically bind thereto).In certain embodiments, the pharmaceutical compositions may also includeone or more agents (e.g., antibodies, antigen binding regions, orsoluble receptors) that specifically bind and inhibit the activity ofgrowth factors, such as antagonists of hepatocyte growth factor (HGF,also known as Scatter Factor), and antibodies or antigen binding regionsthat specifically bind its receptor “c-met.”

Exemplary anti-angiogenic agents include, but are not limited to,Campath, IL-8, B-FGF, Tek antagonists (Ceretti et al., U.S. PatentApplication Publication No. 2003/0162712; U.S. Pat. No. 6,413,932);anti-TWEAK agents (e.g., specifically binding antibodies or antigenbinding regions, or soluble TWEAK receptor antagonists; see, e.g.,Wiley, U.S. Pat. No. 6,727,225); ADAM disintegrin domain to antagonizethe binding of integrin to its ligands (Fanslow et al., U.S. PatentApplication Publication No. 2002/0042368); specifically binding anti-ephreceptor and/or anti-ephrin antibodies or antigen binding regions (U.S.Pat. Nos. 5,981,245; 5,728,813; 5,969,110; 6,596,852; 6,232,447;6,057,124; and patent family members thereof); anti-PDGF-BB antagonists(e.g., specifically binding antibodies or antigen binding regions) aswell as antibodies or antigen binding regions specifically binding toPDGF-BB ligands, and PDGFR kinase inhibitory agents (e.g., antibodies orantigen binding regions that specifically bind thereto).

Exemplary anti-angiogenic/anti-tumor agents include, but are not limitedto, SF-7784 (Pfizer, USA); cilengitide (Merck KgaA, Germany, EPO770622); pegaptanib octasodium (Gilead Sciences, USA); Alphastatin(BioActa, UK); M-PGA (Celgene, USA, U.S. Pat. No. 5,712,291); ilomastat(Arriva, USA, U.S. Pat. No. 5,892,112); emaxanib (Pfizer, USA, U.S. Pat.No. 5,792,783); vatalanib (Novartis, Switzerland); 2-methoxyestradiol(EntreMed, USA); TLC ELL-12 (Elan, Ireland); anecortave acetate (Alcon,USA); alpha-D148 Mab (Amgen, USA); CEP-7055 (Cephalon, USA); anti-Vn Mab(Crucell, Netherlands); DAC:antiangiogenic (ConjuChem, Canada);Angiocidin (InKine Pharmaceutical, USA); KM-2550 (Kyowa Hakko, Japan);SU-0879 (Pfizer, USA); CGP-79787 (Novartis, Switzerland, EP 970070);ARGENT technology (Ariad, USA); YIGSR-Strealth (Johnson & Johnson, USA);fibrinogen-E fragment (BioActa, UK); angiogenesis inhibitor (Trigen,UK); TBC-1635 (Encysive Pharmaceuticals, USA); SC-236 (Pfizer, USA);ABT-567 (Abbott, USA); Metastatin (EntreMed, USA); angiogenesisinhibitor (Tripep, Sweden); maspin (Sosei, Japan); 2-methoxyestradiol(Oncology Sciences Corporation, USA); ER-68203-00 (IVAX, USA); Benefin(Lane Labs, USA); Tz-93 (Tsumura, Japan); TAN-1120 (Takeda, Japan);FR-111142 (Fujisawa, Japan, JP 02233610); platelet factor 4 (RepliGen,USA, EP 407122); vascular endothelial growth factor antagonist (Borean,Denmark); temsirolimus (CCI-779) (University of South Carolina, USA);bevacizumab (pINN) (Genentech, USA); angiogenesis inhibitors (SUGEN,USA); XL 784 (Exelixis, USA); XL 647 (Exelixis, USA); Mab, alpha5beta3integrin, Vitaxin and second generation Vitaxin (Applied MolecularEvolution, USA and MedImmune USA); Retinostat® gene therapy (OxfordBioMedica, UK); enzastaurin hydrochloride (USAN) (Lilly, USA); CEP 7055(Cephalon, USA and Sanofi-Synthelabo, France); BC 1 (Genoa Institute ofCancer Research, Italy); angiogenesis inhibitor (Alchemia, Australia);VEGF antagonist (Regeneron, USA); rBPI 21 and BPI-derived antiangiogenic(XOMA, USA); PI 88 (Progen, Australia); cilengitide (pINN) (Merck KgaA,Germany; Munich Technical University, Germany; Scripps Clinic andResearch Foundation, USA); cetuximab (INN) (Aventis, France); AVE 8062(Ajinomoto, Japan); AS 1404 (Cancer Research Laboratory, New Zealand);SG 292 (Telios, USA); Endostatin (Boston Children's Hospital, USA);2-methoxyestradiol (Boston Childrens Hospital, USA); ZD 6474(AstraZeneca, UK); ZD 6126 (Angiogene Pharmaceuticals, UK); PPI 2458(Praecis, USA); AZD 9935 (AstraZeneca, UK); AZD 2171 (AstraZeneca, UK);vatalanib (pINN) (Novartis, Switzerland and Schering AG, Germany);tissue factor pathway inhibitors (EntraMed, USA); pegaptanib (Pinn)(Gilead Sciences, USA); xanthorrhizol (Yonsei University, South Korea);vaccine, gene-based, VEGF-2 (Scripps Clinic and Research Foundation,USA); SPV5.2 (Supratek, Canada); SDX 103 (University of California atSan Diego, USA); PX 478 (Pro1X, USA); Metastatin (EntreMed, USA);troponin I (Harvard University, USA); SU 6668 (SUGEN, USA); OXI 4503(OXiGENE, USA); o-guanidines (Dimensional Pharmaceuticals, USA);motuporamine C (British Columbia University, Canada); CDP 791 (CelltechGroup, UK); atiprimod (pINN) (GlaxoSmithKline, UK); E 7820 (Eisai,Japan); CYC 381 (Harvard University, USA); AE 941 (Aeterna, Canada);FGF2 cancer vaccine (EntreMed, USA); urokinase plasminogen activatorinhibitor (Dendreon, USA); oglufanide (pINN) (Melmotte, USA); HIF-1alfainhibitors (Xenova, UK); CEP 5214 (Cephalon, USA); BAY RES 2622 (Bayer,Germany); Angiocidin (InKine, USA); A6 (Angstrom, USA); KR 31372 (KoreanResearch Institute of Chemical Technology, South Korea); GW 2286(GlaxoSmithKline, UK); EHT 0101 (ExonHit, France); CP 868596 (Pfizer,USA); CP 564959 (OSI, USA); CP 547632 (Pfizer, USA); 786034(GlaxoSmithKline, UK); KRN 633 (Kirin Brewery, Japan); drug deliverysystem, intraocular, 2-methoxyestradiol (EntreMed, USA); anginex(Maastricht University, Netherlands, and Minnesota University, USA); ABT510 (Abbott, USA); AAL 993 (Novartis, Switzerland); VEGI (ProteomTech,USA); tumor necrosis factor-alpha inhibitors (National Institute onAging, USA); SU 11248 (Pfizer, USA and SUGEN USA); ABT 518 (Abbott,USA); YH16 (Yantai Rongchang, China); S-3APG (Boston Childrens Hospital,USA and EntreMed, USA); Mab, KDR (ImClone Systems, USA); Mab, alpha5beta1 (Protein Design, USA); KDR kinase inhibitor (Celltech Group, UK,and Johnson & Johnson, USA); GFB 116 (South Florida University, USA andYale University, USA); CS 706 (Sankyo, Japan); combretastatin A4 prodrug(Arizona State University, USA); chondroitinase AC (IBEX, Canada); BAYRES 2690 (Bayer, Germany); AGM 1470 (Harvard University, USA, Takeda,Japan, and TAP, USA); AG 13925 (Agouron, USA); Tetrathiomolybdate(University of Michigan, USA); GCS 100 (Wayne State University, USA); CV247 (Ivy Medical, UK); CKD 732 (Chong Kun Dang, South Korea); Mab,vascular endothelium growth factor (Xenova, UK); irsogladine (INN)(Nippon Shinyaku, Japan); RG 13577 (Aventis, France); WX 360 (Wilex,Germany); squalamine (pINN) (Genaera, USA); RPI 4610 (Sirna, USA);galacto fucan sulphate (Marinova, Australia); heparanase inhibitors(InSight, Israel); KL 3106 (Kolon, South Korea); Honokiol (EmoryUniversity, USA); ZK CDK (Shering AG, Germany); ZK Angio (Schering AG,Germany); ZK 229561 (Novartis, Switzerland, and Schering AG, Germany);XMP 300 (XOMA, USA); VGA 1102 (Taisho, Japan); VEGF receptor modulators(Pharmacopeia, USA); VE-cadherin-2 antagonists (ImClone Systems, USA);Vasostatin (National Institutes of Health, USA); vaccine, Flk-1 (ImCloneSystems, USA); TZ 93 (Tsumura, Japan); TumStatin (Beth Israel Hospital,USA); truncated soluble FLT 1 (vascular endothelial growth factorreceptor 1) (Merck & Co, USA); Tie-2 ligands (Regeneron, USA); andthrombospondin 1 inhibitor (Allegheny Health, Education and ResearchFoundation, USA).

Certain cancer therapy agents include, but are not limited to:thalidomide and thalidomide analogues(N-(2,6-dioxo-3-piperidyl)phthalimide); tecogalan sodium (sulfatedpolysaccharide peptidoglycan); Velcade®; bortezomib; rapamycin; TAN 1120(8-acetyl-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-10-[[octahydro-5-hydroxy-2-(2-hydroxypropyl)-4,10-dimethylpyrano[3,4-d]-1,3,6-dioxazocin-8-yl]oxy]-5,12-naphthacenedione);suradista(7,7′-[carbonylbis[imino(1-methyl-1H-pyrrole-4,2-diyl)carbonylimino(1-methyl-1H-pyrrole-4,2-diyl)carbonylimino]]bis-1,3-naphthalenedisulfonicacid tetrasodium salt); SU 302; SU 301; SU 1498((E)-2-cyano-3-[4-hydroxy-3,5-bis(1-methylethyl)phenyl]-N-(3-phenylpropyl)-2-propenamide); SU 1433 (4-(6,7-dimethyl-2-quinoxalinyl)-1,2-benzenediol); ST1514; SR 25989; soluble Tie-2; SERM derivatives; Pharmos; semaxanib(pINN)(3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylene]-1,3-dihydro-2H-indol-2-one);S 836; RG 8803; RESTIN; R 440(3-(1-methyl-1H-indol-3-yl)-4-(1-methyl-6-nitro-1H-indol-3-yl)-1H-pyrrole-2,5-dione);R 123942(1-[6-(1,2,4-thiadiazol-5-yl)-3-pyridazinyl]-N-[3-(trifluoromethyl)phenyl]-4-piperidinamine);prolyl hydroxylase inhibitor; progression elevated genes; prinomastat(INN)((S)-2,2-dimethyl-4-[[p-(4-pyridyloxy)phenyl]sulphonyl]-3-thiomorpholinecarbohydroxamicacid); NV 1030; NM 3(8-hydroxy-6-methoxy-alpha-methyl-1-oxo-1H-2-benzopyran-3-acetic acid);NF 681; NF 050; MIG; METH 2; METH 1; manassantin B(alpha-[1-[4-[5-[4-[2-(3,4-dimethoxyphenyl)-2-hydroxy-1-methylethoxy]-3-methoxyphenyl]tetrahydro-3,4-dimethyl-2-furanyl]-2-methoxyphenoxy]ethyl]-1,3-benzodioxole-5-methanol);KDR monoclonal antibody; alpha5beta3 integrin monoclonal antibody; LY290293 (2-amino-4-(3-pyridinyl)-4H-naphtho[1,2-b]pyran-3-carbonitrile);KP 0201448; KM 2550; integrin-specific peptides; INGN 401; GYKI 66475;GYKI 66462; greenstatin (101-354-plasminogen (human)); gene therapy forrheumatoid arthritis, prostate cancer, ovarian cancer, glioma,endostatin, colorectal cancer, ATF BTPI, antiangiogenesis genes,angiogenesis inhibitor, or angiogenesis; gelatinase inhibitor, FR 111142(4,5-dihydroxy-2-hexanoic acid5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2.5]oct-6-ylester); forfenimex (pINN)(S)-alpha-amino-3-hydroxy-4-(hydroxymethyl)benzeneacetic acid);fibronectin antagonist(1-acetyl-L-prolyl-L-histidyl-L-seryl-L-cystinyl-L-aspartamide);fibroblast growth factor receptor inhibitor; fibroblast growth factorantagonist; FCE 27164(7,7′-[carbonylbis[imino(1-methyl-1H-pyrrole-4,2-diyl)carbonylimino(1-methyl-1H-pyrrole-4,2-diyl)carbonylimino]]bis-1,3,5-naphthalenetrisulfonicacid hexasodium salt); FCE 26752(8,8′-[carbonylbis[imino(1-methyl-1H-pyrrole-4,2-diyl)carbonylimino(1-methyl-1H-pyrrole-4,2-diyl)carbonylimino]]bis-1,3,6-naphthalenetrisulfonicacid); endothelial monocyte activating polypeptide II; VEGFR antisenseoligonucleotide; anti-angiogenic and trophic factors; ANCHOR angiostaticagent; endostatin; Del-1 angiogenic protein; CT 3577; contortrostatin;CM 101; chondroitinase AC; CDP 845; CanStatin; BST 2002; BST 2001; BLS0597; BIBF 1000; ARRESTIN; apomigren (1304-1388-type XV collagen (humangene COL15A1 alpha1-chain precursor)); angioinhibin; aaATIII; A 36;9alpha-fluoromedroxyprogesterone acetate((6-alpha)-17-(acetyloxy)-9-fluoro-6-methyl-pregn-4-ene-3,20-dione);2-methyl-2-phthalimidino-glutaric acid(2-(1,3-dihydro-1-oxo-2H-isoindol-2-yl)-2-methylpentanedioic acid);Yttrium 90 labelled monoclonal antibody BC-1; Semaxanib(3-(4,5-Dimethylpyrrol-2-ylmethylene)indolin-2-one)(C15 H14 N2 O); PI 88(phosphomannopentaose sulfate); Alvocidib (4H-1-Benzopyran-4-one,2-(2-chlorophenyl)-5,7-dihydroxy-8-(3-hydroxy-1-methyl-4-piperidinyl)-cis-(−)-)(C21 H2O Cl N O5); E 7820; SU 11248(5-[3-Fluoro-2-oxo-1,2-dihydroindol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylaminoethyl)amide) (C22 H27 F N4 O2); Squalamine(Cholestane-7,24-diol, 3-[[3-[(4-aminobutyl)aminopropyl]amino]-,24-(hydrogen sulfate), (3.beta., 5.alpha., 7.alpha.)-) (C34 H65 N3O5 S);Eriochrome Black T; AGM 1470 (Carbamic acid, (chloroacetyl)-,5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2,5]oct-6-ylester, [3R-[3alpha, 4alpha(2R,3R), 5beta, 6beta]]) (C19 H28 Cl N O6);AZD 9935; BIBF 1000; AZD 2171; ABT 828; KS-interleukin-2; Uteroglobin; A6; NSC 639366(1-[3-(Diethylamino)-2-hydroxypropylamino]-4-(oxyran-2-ylmethylamino)anthraquinonefumarate) (C24 H29 N3 O4. C4 H4 O4); ISV 616; anti-ED-B fusion proteins;HUI 77; Troponin I; BC-1 monoclonal antibody; SPV 5.2; ER 68203; CKD 731(3-(3,4,5-Trimethoxyphenyl)-2(E)-propenoic acid(3R,4S,5S,6R)-4-[2(R)-methyl-3(R)-3(R)-(3-methyl-2-butenyl)oxiran-2-yl]-5-methoxy-1-oxaspiro[2.5]oct-6-ylester) (C28 H38 O8); IMC-1C11; aaATIII; SC 7; CM 101; Angiocol; Kringle5; CKD 732 (3-[4-[2-(Dimethylamino)ethoxy]phenyl]-2(E)-propenoic acid)(C29 H41 N O6); U 995; Canstatin; SQ 885; CT 2584(1-[11-(Dodecylamino)-10-hydroxyundecyl]-3,7-dimethylxanthine)(C30 H55N5 O3); Salmosin; EMAP II; TX 1920(1-(4-Methylpiperazino)-2-(2-nitro-1H-1-imidazoyl)-1-ethanone) (C10 H15N5 O3); Alpha-v Beta-x inhibitor; CHIR 11509(N-(1-Propynyl)glycyl-[N-(2-naphthyl)]glycyl-[N-(carbamoylmethyl)]glycinebis(4-methoxyphenyl)methylamide)(C36 H37 N5 O6); BST 2002; BST 2001; B0829; FR 111142; 4,5-Dihydroxy-2(E)-hexanoic acid(3R,4S,5S,6R)-4-[1(R),2(R)-epoxy-1,5-dimethyl-4-hexenyl]-5-methoxy-1-oxaspiro[2.5]octan-6-ylester (C22 H34 O7); and kinase inhibitors including, but not limited to,N-(4-chlorophenyl)-4-(4-pyridinylmethyl)-1-phthalazinamine;4-[4-[[[[4-chloro-3-(trifluoromethyl)phenyl]amino]carbonyl]amino]phenoxy]-N-methyl-2-pyridinecarboxamide;N-[2-(diethylamino)ethyl]-5-[(5-fluoro-1,2-dihydro-2-oxo-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide;3-[(4-bromo-2,6-difluorophenyl)methoxy]-5-[[[[4-(1-pyrrolidinyl)butyl]amino]carbonyl]amino]-4-isothiazolecarboxamide;N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methyl-4-piperidinyl)methoxy]-4-quinazolinamine;3-[5,6,7,13-tetrahydro-9-[(1-methylethoxy)methyl]-5-oxo-12H-indeno[2,1-a]pyrrolo[3,4-c]carbazol-12-yl]propylester N,N-dimethyl-glycine;N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide;N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6-[5-[[[2-(methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine;4-[(4-Methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]-phenyl]benzamide;N-(3-chloro-4-fluorophenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamine;N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine;N-(3-((((2R)-1-methyl-2-pyrrolidinyl)methyl)oxy)-5-(trifluoromethyl)phenyl)-2-((3-(1,3-oxazol-5-yl)phenyl)amino)-3-pyridinecarboxamide;2-(((4-fluorophenyl)methyl)amino)-N-(3-((((2R)-1-methyl-2-pyrrolidinyl)methyl)oxy)-5-(trifluoromethyl)phenyl)-3-pyridinecarboxamide;N-[3-(Azetidin-3-ylmethoxy)-5-trifluoromethyl-phenyl]-2-(4-fluoro-benzylamino)-nicotinamide;6-fluoro-N-(4-(1-methylethyl)phenyl)-2-((4-pyridinylmethyl)amino)-3-pyridinecarboxamide;2-((4-pyridinylmethyl)amino)-N-(3-(((2S)-2-pyrrolidinylmethyl)oxy)-5-(trifluoromethyl)phenyl)-3-pyridinecarboxamide;N-(3-(1,1-dimethylethyl)-1H-pyrazol-5-yl)-2-((4-pyridinylmethyl)amino)-3-pyridinecarboxamide;N-(3,3-dimethyl-2,3-dihydro-1-benzofuran-6-yl)-2-((4-pyridinylmethyl)amino)-3-pyridinecarboxamide;N-(3-((((2S)-1-methyl-2-pyrrolidinyl)methyl)oxy)-5-(trifluoromethyl)phenyl)-2-((4-pyridinylmethyl)amino)-3-pyridinecarboxamide;2-((4-pyridinylmethyl)amino)-N-(3-((2-(1-pyrrolidinyl)ethyl)oxy)-4-(trifluoromethyl)phenyl)-3-pyridinecarboxamide;N-(3,3-dimethyl-2,3-dihydro-1H-indol-6-yl)-2-((4-pyridinylmethyl)amino)-3-pyridinecarboxamide;N-(4-(pentafluoroethyl)-3-(((2S)-2-pyrrolidinylmethyl)oxy)phenyl)-2-((4-pyridinylmethyl)amino)-3-pyridinecarboxamide;N-(3-((3-azetidinylmethyl)oxy)-5-(trifluoromethyl)phenyl)-2-((4-pyridinylmethyl)amino)-3-pyridinecarboxamide;N-(3-(4-piperidinyloxy)-5-(trifluoromethyl)phenyl)-2-((2-(3-pyridinyl)ethyl)amino)-3-pyridinecarboxamide;N-(4,4-dimethyl-1,2,3,4-tetrahydro-isoquinolin-7-yl)-2-(1H-indazol-6-ylamino)-nicotinamide;2-(1H-indazol-6-ylamino)-N-[3-(1-methylpyrrolidin-2-ylmethoxy)-5-trifluoromethyl-phenyl]-nicotinamide;N-[1-(2-dimethylamino-acetyl)-3,3-dimethyl-2,3-dihydro-1H-indol-6-yl]-2-(1H-indazol-6-ylamino)-nicotinamide;2-(1H-indazol-6-ylamino)-N-[3-(pyrrolidin-2-ylmethoxy)-5-trifluoromethyl-phenyl]-nicotinamide;N-(1-acetyl-3,3-dimethyl-2,3-dihydro-1H-indol-6-yl)-2-(1H-indazol-6-ylamino)-nicotinamide;N-(4,4-dimethyl-1-oxo-1,2,3,4-tetrahydro-isoquinolin-7-yl)-2-(1H-indazol-6-ylamino)-nicotinamide;N-[4-(tert-butyl)-3-(3-piperidylpropyl)phenyl][2-(1H-indazol-6-ylamino)(3-pyridyl)]carboxamide;N-[5-(tert-butyl) isoxazol-3-yl][2-(1H-indazol-6-ylamino)(3-pyridyl)]carboxamide; andN-[4-(tert-butyl)phenyl][2-(1H-indazol-6-ylamino)(3-pyridyl)]carboxamide,and kinase inhibitors disclosed in U.S. Pat. Nos. 6,258,812; 6,235,764;6,630,500; 6,515,004; 6,713,485; 5,521,184; 5,770,599; 5,747,498;5,990,141; U.S. Patent Application Publication No. US2003/0105091; andPatent Cooperation Treaty publication nos. WO01/37820; WO01/32651;WO02/68406; WO02/66470; WO02/55501; WO04/05279; WO04/07481; WO04/07458;WO04/09784; WO02/59110; WO99/45009; WO98/35958; WO00/59509; WO99/61422;WO00/12089; and WO00/02871, each of which publications are herebyincorporated by reference for any purpose.

TR-2 is expressed in a variety of cells, including liver, brain, kidney,colon, breast, lung, spleen, thymus, peripheral blood lymphocytes,pancreas, prostate, testis, ovary, uterus, and various tissues along thegastro-intestinal tract. Exemplary TR-2 related cancers include, but arenot limited to, liver cancer, brain cancer, renal cancer, breast cancer,pancreatic cancer, colorectal cancer, lung cancer (small cell lungcancer and non-small-cell lung cancer), spleen cancer, cancer of thethymus or blood cells (i.e., leukemia), prostate cancer, testicularcancer, ovarian cancer, uterine cancer, gastric carcinoma, head and necksquamous cell carcinoma, melanoma, and lymphoma.

In certain embodiments, an anti-TR-2 antibody may be used alone or withat least one additional therapeutic agent for the treatment of cancer.In certain embodiments, an anti-TR-2 antibody is used in conjunctionwith a therapeutically effective amount of an additional therapeuticagent. Exemplary therapeutic agents that may be administered with ananti-TR-2 antibody include, but are not limited to, a member of thegeldanamycin family of ansamycin antibiotics; a Pro-HGF; NK2; a c-Metpeptide inhibitor; an antagonist of Grb2 Src homology 2; a Gab1modulator; dominant-negative Src; a von-Hippel-Landau inhibitor,including, but not limited to, wortmanin; P13 kinase inhibitors, otheranti-receptor therapies, anti EGFr, a COX-2 inhibitor, Celebrex™,celecoxib, Vioxx™, rofecoxib; a vascular endothelial growth factor(VEGF), a VEGF modulator, a fibroblast growth factor (FGF), an FGFmodulator, an epidermal growth factor (EGF); an EGF modulator; akeratinocyte growth factor (KGF), a KGF-related molecule, a KGFmodulator; and a matrix metalloproteinase (MMP) modulator.

In certain embodiments, anti-TR-2 antibody is used with particulartherapeutic agents to treat various cancers. In certain embodiments, inview of the condition and the desired level of treatment, two, three, ormore agents may be administered. Where the compounds are used togetherwith one or more other components, the compound and the one or moreother components may be administered together, separately, orsequentially (e.g., in a pharmaceutical format). In certain embodiments,such agents may be provided together by inclusion in the sameformulation. In certain embodiments, such agents and an anti-TR-2antibody may be provided together by inclusion in the same formulation.In certain embodiments, such agents may be formulated separately andprovided together by inclusion in a treatment kit. In certainembodiments, such agents and an anti-TR-2 antibody may be formulatedseparately and provided together by inclusion in a treatment kit. Incertain embodiments, such agents may be provided separately.

In certain embodiments, when administered by gene therapy, the genesencoding protein agents and/or an anti-TR-2 antibody may be included inthe same vector. In certain embodiments, the genes encoding proteinagents and/or an anti-TR-2 antibody may be under the control of the samepromoter region. In certain embodiments, the genes encoding proteinagents and/or an anti-TR-2 antibody may be in separate vectors.

In certain embodiments, anti-TR-2 antibodies may be used to treatnon-human animals, such as pets (dogs, cats, birds, primates, etc.), anddomestic farm animals (horses cattle, sheep, pigs, birds, etc.). Incertain such instances, an appropriate dose may be determined accordingto the animal's body weight. For example, in certain embodiments, a doseof 0.2-1 mg/kg may be used. In certain embodiments, the dose may bedetermined according to the animal's surface area, an exemplary doseranging from 0.1 to 20 mg/in², or from 5 to 12 mg/m². For small animals,such as dogs or cats, in certain embodiments, a suitable dose is 0.4mg/kg. In certain embodiments, anti-TR-2 antibodies are administered byinjection or other suitable route one or more times per week until theanimal's condition is improved, or it may be administered indefinitely.

It is understood that the response by individual patients to theaforementioned medications or combination therapies may vary, and anappropriate efficacious combination of drugs for each patient may bedetermined by his or her physician.

The cynomolgus monkey provides a useful model for certain diseases.Exemplary diseases include, but are not limited to, transplantationrejection syndrome and inflammatory bowel disease-like disease. Whentesting the efficacy of a human MAb in cynomolgus monkey human diseasemodel, in certain embodiments, it is useful to determine whether theanti-TR-2 MAb binds to TR-2 in humans and cynomolgus monkeys at acomparable level.

In certain embodiments, an anti-TR-2 antibody may be part of a conjugatemolecule comprising all or part of the anti-TR-2 antibody and acytotoxic agent. The term “cytotoxic agent” refers to a substance thatinhibits or prevents the function of cells and/or causes the death ordestruction of cells. The term includes, but is not limited to,radioactive isotopes (e.g., I¹³¹, I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeuticagents, and toxins such as enzymatically active toxins of bacterial,fungal, plant or animal origin, or fragments thereof. Exemplarycytotoxic agents include, but are not limited to, Adriamycin,Doxorubicin, 5-Fluorouracil, Cytosine arabinoside (“Ara-C”),Cyclophosphamide, Thiotepa, Taxotere (docetaxel), Busulfan, Cytoxin,Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin,Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincristine,Vinorelbine, Carboplatin, Teniposide, Daunomycin, Caminomycin,Aminopterin, Dactinomycin, Mitomycins, Esperamicins, Melphalan and otherrelated nitrogen mustards.

In certain embodiments, an anti-TR-2 antibody may be part of a conjugatemolecule comprising all or part of the anti-TR-2 antibody and a prodrug.In certain embodiments, the term “prodrug” refers to a precursor orderivative form of a pharmaceutically active substance. In certainembodiments, a prodrug is less cytotoxic to cells compared to the parentdrug and is capable of being enzymatically activated or converted intothe more active cytotoxic parent form. Exemplary prodrugs include, butare not limited to, phosphate-containing prodrugs,thiophosphate-containing prodrugs, sulfate-containing prodrugs,peptide-containing prodrugs, D-amino acid-modified prodrugs,glycosylated prodrugs, beta-lactam-containing prodrugs, optionallysubstituted phenoxyacetamide-containing prodrugs and optionallysubstituted phenylacetamide-containing prodrugs, 5-fluorocytosine andother 5-fluorouridine prodrugs which can be converted into a more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form include, but are not limited to, those cytotoxicagents described above. See, e.g., U.S. Pat. No. 6,702,705.

In certain embodiments, antibody conjugates function by having theantibody portion of the molecule target the cytotoxic portion or prodrugportion of the molecule to a specific population of cells in thepatient. In the case of anti-TR-2 antibodies, such conjugate moleculesmay be used, for example, in certain embodiments, to destroy abnormallyproliferating cells, such as cancer cells.

In certain embodiments, methods of treating a patient comprisingadministering a therapeutically effective amount of an anti-TR-2antibody are provided. In certain embodiments, methods of treating apatient comprising administering a therapeutically effective amount ofan antibody conjugate are provided. In certain embodiments, an antibodyis used in conjunction with a therapeutically effective amount of atleast one additional therapeutic agent, as discussed above.

As discussed above, in certain embodiments, anti-TR-2 antibodies may beadministered concurrently with one or more other drugs that areadministered to the same patient, each drug being administered accordingto a regimen suitable for that medicament. Such treatment encompassespre-treatment, simultaneous treatment, sequential treatment, andalternating regimens. Additional examples of such drugs include, but arenot limited to, antivirals, antibiotics, analgesics, corticosteroids,antagonists of inflammatory cytokines, DMARDs, nonsteroidalanti-inflammatories, chemotherapeutics, inhibitors of angiogenesis, andstimulators of angiogenesis.

In certain embodiments, various medical disorders are treated withanti-TR-2 antibodies in combination with another stimulator ofapoptosis. For example, in certain embodiments, anti-TR-2 antibodies maybe administered in a composition that also contains a compound thatstimulates apoptosis of one or more cells. In certain embodiments, theanti-TR-2 antibody and stimulators of apoptosis may be administered asseparate compositions, and these may be administered by the same ordifferent routes.

In certain embodiments, pharmaceutical compositions are providedcomprising a therapeutically effective amount of an antibody togetherwith a pharmaceutically acceptable diluent, carrier, solubilizer,emulsifier, preservative and/or adjuvant.

In certain embodiments, pharmaceutical compositions are providedcomprising a therapeutically effective amount of an antibody and atherapeutically effective amount of at least one additional therapeuticagent, together with a pharmaceutically acceptable diluent, carrier,solubilizer, emulsifier, preservative and/or adjuvant.

In certain embodiments, acceptable formulation materials preferably arenontoxic to recipients at the dosages and concentrations employed. Incertain embodiments, antibodies of the present invention are provided ina bufferless formulation as disclosed in PCT/US06/22599 filed Jun. 8,2006, which is incorporated by reference herein for any purpose.

In certain embodiments, the pharmaceutical composition may containformulation materials for modifying, maintaining or preserving, forexample, the pH, osmolarity, viscosity, clarity, color, isotonicity,odor, sterility, stability, rate of dissolution or release, adsorptionor penetration of the composition. In certain embodiments, suitableformulation materials include, but are not limited to, amino acids (suchas glycine, glutamine, asparagine, arginine or lysine); antimicrobials;antioxidants (such as ascorbic acid, sodium sulfite or sodiumhydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl,citrates, phosphates or other organic acids); bulking agents (such asmannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate 80, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants.(Remington's Pharmaceutical Sciences, 18^(th) Edition, A. R. Gennaro,ed., Mack Publishing Company (1990).

In certain embodiments, an antibody and/or an additional therapeuticmolecule is linked to a half-life extending vehicle known in the art.Such vehicles include, but are not limited to, the Fc domain,polyethylene glycol, and dextran. Such vehicles are described, e.g., inU.S. Pat. No. 6,660,843 and published PCT Application No. WO 99/25044.

In certain embodiments, the optimal pharmaceutical composition will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, Remington's Pharmaceutical Sciences, supra. In certainembodiments, such compositions may influence the physical state,stability, rate of in vivo release and rate of in vivo clearance of theantibodies.

In certain embodiments, the primary vehicle or carrier in apharmaceutical composition may be either aqueous or non-aqueous innature. For example, in certain embodiments, a suitable vehicle orcarrier may be water for injection, physiological saline solution orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Incertain embodiments, neutral buffered saline or saline mixed with serumalbumin are further exemplary vehicles. In certain embodiments,pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, oracetate buffer of about pH 4.0-5.5, which may further include sorbitolor a suitable substitute therefor. In certain embodiments, apharmaceutical composition is an aqueous or liquid formulationcomprising an acetate buffer of about pH 4.0-5.5, a polyol(polyalcohol), and optionally, a surfactant, wherein the compositiondoes not comprise a salt, e.g., sodium chloride, and wherein thecomposition is isotonic for the patient. Exemplary polyols include, butare not limited to, sucrose, glucose, sorbitol, and mannitol. Anexemplary surfactant includes, but is not limited to, polysorbate. Incertain embodiments, a pharmaceutical composition is an aqueous orliquid formulation comprising an acetate buffer of about pH 5.0,sorbitol, and a polysorbate, wherein the composition does not comprise asalt, e.g., sodium chloride, and wherein the composition is isotonic forthe patient. Certain exemplary compositions are found, for example, inU.S. Pat. No. 6,171,586. Additional pharmaceutical carriers include, butare not limited to, oils, including petroleum oil, animal oil, vegetableoil, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Incertain embodiments, aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions. Incertain embodiments, a composition comprising an antibody, with orwithout at least one additional therapeutic agent, may be prepared forstorage by mixing the selected composition having the desired degree ofpurity with optional formulation agents (Remington's PharmaceuticalSciences, supra) in the form of a lyophilized cake or an aqueoussolution. Further, in certain embodiments, a composition comprising anantibody, with or without at least one additional therapeutic agent, maybe formulated as a lyophilizate using appropriate excipient solutions(e.g., sucrose) as diluents.

In certain embodiments, anti-TR-2 antibodies are administered in theform of a physiologically acceptable composition comprising purifiedrecombinant protein in conjunction with physiologically acceptablecarriers, excipients or diluents. In certain embodiments, such carriersare nontoxic to recipients at the dosages and concentrations employed.In certain embodiments, preparing such compositions may involvecombining the anti-TR-2 antibodies with buffers, antioxidants such asascorbic acid, low molecular weight polypeptides (such as those havingfewer than 10 amino acids), proteins, amino acids, carbohydrates such asglucose, sucrose or dextrins, chelating agents such as EDTA, glutathioneand/or other stabilizers, and excipients. In certain embodiments,appropriate dosages are determined in standard dosing trials, and mayvary according to the chosen route of administration. In certainembodiments, in accordance with appropriate industry standards,preservatives may also be added, which include, but are not limited to,benzyl alcohol. In certain embodiments, the amount and frequency ofadministration may be determined based on such factors as the nature andseverity of the disease being treated, the desired response, the age andcondition of the patient, and so forth.

In certain embodiments, pharmaceutical compositions can be selected forparenteral delivery. The preparation of certain such pharmaceuticallyacceptable compositions is within the skill of the art.

In certain embodiments, the formulation components are present inconcentrations that are acceptable to the site of administration. Incertain embodiments, buffers are used to maintain the composition atphysiological pH or at a slightly lower pH, typically within a pH rangeof from about 5 to about 8.

In certain embodiments, when parenteral administration is contemplated,a therapeutic composition may be in the form of a pyrogen-free,parenterally acceptable aqueous solution comprising the desiredantibody, with or without additional therapeutic agents, in apharmaceutically acceptable vehicle. In certain embodiments, a vehiclefor parenteral injection is sterile distilled water in which theantibody, with or without at least one additional therapeutic agent, isformulated as a sterile, isotonic solution, properly preserved. Incertain embodiments, the preparation can involve the formulation of thedesired molecule with an agent, such as injectable microspheres,bio-erodible particles, polymeric compounds (such as polylactic acid orpolyglycolic acid), beads, or liposomes, that may provide for thecontrolled or sustained release of the product which may then bedelivered via a depot injection. In certain embodiments, hyaluronic acidmay also be used, and may have the effect of promoting sustainedduration in the circulation. In certain embodiments, implantable drugdelivery devices may be used to introduce the desired molecule.

In certain embodiments, a pharmaceutical composition may be formulatedfor inhalation. In certain embodiments, an antibody, with or without atleast one additional therapeutic agent, may be formulated as a drypowder for inhalation. In certain embodiments, an inhalation solutioncomprising an antibody, with or without at least one additionaltherapeutic agent, may be formulated with a propellant for aerosoldelivery. In certain embodiments, solutions may be nebulized. Pulmonaryadministration is further described in PCT publication no. WO94/20069,which describes pulmonary delivery of chemically modified proteins.

In certain embodiments, it is contemplated that formulations may beadministered orally. In certain embodiments, an antibody, with orwithout at least one additional therapeutic agent, that is administeredin this fashion may be formulated with or without those carrierscustomarily used in the compounding of solid dosage forms such astablets and capsules. In certain embodiments, a capsule may be designedto release the active portion of the formulation at the point in thegastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. In certain embodiments, at leastone additional agent can be included to facilitate absorption of theantibody and/or any additional therapeutic agents. In certainembodiments, diluents, flavorings, low melting point waxes, vegetableoils, lubricants, suspending agents, tablet disintegrating agents,and/or binders may also be employed.

In certain embodiments, a pharmaceutical composition may involve aneffective quantity of antibodies, with or without at least oneadditional therapeutic agent, in a mixture with non-toxic excipientswhich are suitable for the manufacture of tablets. In certainembodiments, by dissolving the tablets in sterile water, or anotherappropriate vehicle, solutions may be prepared in unit-dose form.Suitable excipients include, but are not limited to, inert diluents,such as calcium carbonate, sodium carbonate or bicarbonate, lactose, orcalcium phosphate; and binding agents, such as starch, gelatin, andacacia; and lubricating agents such as magnesium stearate, stearic acid,and talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving antibodies, with or withoutat least one additional therapeutic agent, in sustained- orcontrolled-delivery formulations. In certain exemplary sustained- orcontrolled-delivery formulations include, but are not limited to,liposome carriers, bio-erodible microparticles, porous beads, and depotinjections. Certain exemplary techniques for preparing certainformulations are known to those skilled in the art. See for example, PCTpublication no. WO93/15722, which describes the controlled release ofporous polymeric microparticles for the delivery of pharmaceuticalcompositions. In certain embodiments, sustained-release preparations mayinclude semipermeable polymer matrices in the form of shaped articles,e.g. films, or microcapsules. Sustained release matrices include, butare not limited to, polyesters, hydrogels, polylactides (U.S. Pat. No.3,773,919 and EP 058,481), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., Biopolymers, 22:547-556 (1983)),poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater.Res., 15:167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)),ethylene vinyl acetate (Langer et al., supra), andpoly-D(−)-3-hydroxybutyric acid (EP 133,988). In certain embodiments,sustained release compositions may also include liposomes, which can beprepared, in certain embodiments, by any of several methods known in theart. See e.g., Eppstein et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692(1985); EP 036,676; EP 088,046 and EP 143,949.

In certain embodiments, the pharmaceutical composition to be used for invivo administration is sterile. In certain embodiments, thepharmaceutical composition to be used for in vivo administration is madesterile by filtration through sterile filtration membranes. In certainembodiments, where the composition is lyophilized, sterilization usingsterile filtration membranes may be conducted either prior to orfollowing lyophilization and reconstitution. In certain embodiments, thecomposition for parenteral administration may be stored in lyophilizedform or in a solution. In certain embodiments, parenteral compositionsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

In certain embodiments, after the pharmaceutical composition has beenformulated, it may be stored in sterile vials as a solution, suspension,gel, emulsion, solid, or as a dehydrated or lyophilized powder. Incertain embodiments, such formulations may be stored either in aready-to-use form or in a form (e.g., a lyophilized form) that isreconstituted prior to administration.

In certain embodiments, kits for producing a single-dose administrationunit are provided. In certain embodiments, the kits may each containboth a first container having a dried protein and a second containerhaving an aqueous formulation. In certain embodiments, kits containingsingle and/or multi-chambered pre-filled syringes (e.g., liquid syringesand lyosyringes) are included.

In certain embodiments, the effective amount of a pharmaceuticalcomposition comprising an antibody, with or without at least oneadditional therapeutic agent, to be employed therapeutically willdepend, for example, upon the therapeutic context and objectives. Oneskilled in the art will appreciate that the appropriate dosage levelsfor treatment, according to certain embodiments, will thus varydepending, in part, upon the molecule delivered, the indication forwhich the antibody, with or without at least one additional therapeuticagent, is being used, the route of administration, and the size (bodyweight, body surface or organ size) and/or condition (the age andgeneral health) of the patient. In certain embodiments, the clinicianmay titer the dosage and modify the route of administration to obtainthe optimal therapeutic effect. In certain embodiments, a typical dosagemay range from about 0.1 μg/kg to up to about 100 mg/kg or more,depending on the factors mentioned above. In certain embodiments, thedosage may range from 0.1 μg/kg up to about 100 mg/kg; or 1 μg/kg up toabout 100 mg/kg; or 5 μg/kg up to about 100 mg/kg; or 0.1 mg/kg up toabout 100 mg/kg.

In certain embodiments, the frequency of dosing will take into accountthe pharmacokinetic parameters of the antibody and/or any additionaltherapeutic agents in the formulation used. In certain embodiments, aclinician will administer the composition until a dosage is reached thatachieves the desired effect. In certain embodiments, the composition maytherefore be administered as a single dose, or as two or more doses(which may or may not contain the same amount of the desired molecule)over time, or as a continuous infusion via an implantation device orcatheter. Certain methods of further refining the appropriate dosage arewithin the skill in the art. In certain embodiments, appropriate dosagesmay be ascertained through use of appropriate dose-response data.

In certain embodiments, the route of administration of thepharmaceutical composition is in accord with known methods, e.g. orally,through injection by intravenous, intraperitoneal, intracerebral(intra-parenchymal), intracerebroventricular, intramuscular,intra-ocular, intraarterial, intraportal, or intralesional routes; bysustained release systems or by implantation devices. In certainembodiments, the compositions may be administered by bolus injection orcontinuously by infusion, or by implantation device.

As discussed above, in various embodiments, any efficacious route ofadministration may be used to administer anti-TR-2 antibodies. Ifinjected, in certain embodiments, anti-TR-2 antibodies may beadministered, for example, via intra-articular, intravenous,intramuscular, intralesional, intraperitoneal, intracranial, intranasal,inhalation or subcutaneous routes by bolus injection or by continuousinfusion. Exemplary methods of administration include, but are notlimited to, sustained release from implants, aerosol inhalation,eyedrops, oral preparations, including pills, syrups, lozenges, andchewing gum, and topical preparations such as lotions, gels, sprays,ointments, and other suitable techniques.

In certain embodiments, administration by inhalation is beneficial whentreating diseases associated with pulmonary disorders. In certainembodiments, anti-TR-2 antibodies may be administered by implantingcultured cells that express the antibodies. In certain embodiments, thepatient's own cells are induced to produce by transfection in vivo or exvivo with one or more vectors that encode an anti-TR-2 antibody. Incertain embodiments, this vector can be introduced into the patient'scells, for example, by injecting naked DNA or liposome-encapsulated DNAthat encodes an anti-TR-2 antibody, or by other methods of transfection.When anti-TR-2 antibodies are administered in combination with one ormore other biologically active compounds, in certain embodiments, thesemay be administered by the same or by different routes, and may beadministered together, separately, or sequentially.

In certain embodiments, the composition may be administered locally viaimplantation of a membrane, sponge or another appropriate material ontowhich the desired molecule has been absorbed or encapsulated. In certainembodiments, where an implantation device is used, the device may beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule may be via diffusion, timed-release bolus, or continuousadministration.

In certain embodiments, it may be desirable to use a pharmaceuticalcomposition comprising an antibody, with or without at least oneadditional therapeutic agent, in an ex vivo manner. In such embodiments,cells, tissues and/or organs that have been removed from the patient areexposed to a pharmaceutical composition comprising an antibody, with orwithout at least one additional therapeutic agent, after which thecells, tissues and/or organs are subsequently implanted back into thepatient.

In certain embodiments, an antibody and any additional therapeuticagents can be delivered by implanting certain cells that have beengenetically engineered, using methods such as those described herein, toexpress and secrete the polypeptides. In certain embodiments, such cellsmay be animal or human cells, and may be autologous, heterologous, orxenogeneic. In certain embodiments, the cells may be immortalized. Incertain embodiments, in order to decrease the chance of an immunologicalresponse, the cells may be encapsulated to avoid infiltration ofsurrounding tissues. In certain embodiments, the encapsulation materialsare typically biocompatible, semi-permeable polymeric enclosures ormembranes that allow the release of the protein product(s) but preventthe destruction of the cells by the patient's immune system or by otherdetrimental factors from the surrounding tissues.

EXAMPLES Example 1 Production of Certain Human Monoclonal Antibodies

Human anti-TR-2 antibodies were produced in one of two ways. Transgenicmice expressing human immunoglobulin genes (Xenomouse®) were exposed tohuman TR-2. Certain human anti-TR-2 monoclonal antibodies were producedfrom those mice using hybridoma techniques. Certain other humananti-TR-2 monoclonal antibodies were produced from those mice usingXenoMax technology, which incorporates the selected lymphocyte antibodymethod (“SLAM”) technique (see, e.g., U.S. Pat. No. 5,627,052; andBabcook et al., Proc. Natl. Acad. Sci. USA 93:7843-7848 (1996)).

The methodology used to produce human anti-TR-2 monoclonal antibodies intransgenic mice expressing human immunoglobulin genes was as follows.Five groups of mice were immunized with recombinant human TR-2 with aC-terminal hexahistidine tag (TR-2-His) (mature amino acid sequenceALITQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKESGTKHSGEAPAVEETVT SSPGTPASRSGSSHHHHHH(SEQ ID NO: 140)) (Genbank Reference Number NM-003842), as shown inFIG. 1. The mice in group one, group three, group four, and group fivewere engineered to produce antibodies of the IgG2 isotype (FIG. 2). Themice in group two were engineered to produce antibodies of the IgG4isotype (FIG. 2). Group one included 7 mice, group two included 8 mice,group three included 8 mice, group four included 10 mice, and group fiveincluded 5 mice. The mice in group one, group two, and group three wereimmunized by injection of TR-2-His into the footpad (10 μg perinjection), while the mice in group four and group five were immunizedintraperitoneally (10 μg per injection) with TR-2-His. On day 0, 10 μgantigen was administered by the described route. At specified intervals,booster injections were administered to the mice. Group one mice hadnine booster injections, at days 5, 11, 14, 18, 24, 28, 34, 42, and 46.Group two and group three mice had 7 booster injections; those for group2 were at days 3, 7, 10, 14, 17, 24, and 27, and those for group threewere at days 5, 8, 15, 21, 26, 30, and 33. Group four and group fivemice had 5 booster injections, at days 14, 28, 42, 56, and 72. Eachfirst injection and each booster injection contained 10 μg TR-2-His withan adjuvant, either Titermax Gold (Groups one, two, and three), alum gel(groups one, two, and three), Complete Freund's Adjuvant (CFA) (groupsfour and five), Incomplete Freund's Adjuvant (IFA) (groups four andfive), or Dulbecco's phosphate-buffered saline (D-PBS) (groups one, two,three, four, and five) (see FIG. 1). Mice were bled after threeinjections (groups four and five), after four injections (groups one,two, and three), after six injections (groups one and two), and afterten injections (group one). The reactivity of each bleed to TR-2-His wasassessed by ELISA, as shown in FIG. 2.

The ELISA assay was performed as follows. Multiwell plates were coatedwith soluble TR-2-His (0.5 μg/mL) by passive adsorption overnight at 4°C. The coated wells were washed and blocked for 30 minutes with milk.Ten μL of each mouse serum was combined with 40 μL milk and incubated inthe wells of different plates for 1 hour, 1.25 hours, or 2 hours. Theplates were washed five times with water. The plates were then incubatedwith a goat anti-human IgG Fc-specific horseradish peroxidase-conjugatedantibody (Pierce) at a final concentration of 1 μg/mL for 1 hour at roomtemperature. The plates were washed five times with water. The plateswere incubated with K blue substrate (Neogen) for 30 minutes. Negativecontrols included blank wells lacking TR-2-His and wells includingTR-2-His but incubated with naive G2 sera expected to lack anti-TR-2antibodies.

The methodologies used to produce human anti-TR-2 monoclonal antibodieswere as follows. For XenoMax technology, CD19+ B cells were isolatedfrom the hyperimmune transgenic mice that were harvested on day 37(mouse M712-7 from group three), or day 76 (mouse M564-1 from groupfour, and mice M564-3, M564-5, and M563-5 from group five after theinitiation of immunization. The B cells were cultured for 1 week toallow their expansion and consequent differentiation into plasma cells.The supernatants containing the secreted antibody was saved for furtheranalysis and the plasma cells in each well were frozen at −80 degreescelcius in media containing 10% DMSO and 90% FCS. For hybridomatechnology, the cells from the remaining hyperimmune transgenic micewere harvested on day 31, 37 or 46 for further analysis as shown in FIG.1.

For XenoMax technology, supernates from the B cell cultures werescreened by ELISA for the presence of antibodies to TR-2. Anti-TR-2antibodies were detected by assessing binding to immobilized TR-2-Hisusing an anti-human IgG antibody detection reagent as follows. Plateswere coated with soluble TR-2-His (0.5 μg/ml) by passive adsorptionovernight at 4° C. After washing the plates five times with water andblocking the wells in the plates with milk for 30 minutes, 10 μL cellculture supernate from each individual hybridoma was combined with 40 μLmilk and incubated in the wells of different plates for 1 hour, 1.25hours, or 2 hours. The plates were washed five times with water, andincubated with a goat anti-human IgG Fc-specific horseradish peroxidase(Pierce)-conjugated antibody at a final concentration of 1 μg/mL for 1hour at room temperature. After washing the plates five times withwater, the plates were incubated with K blue substrate (Neogen) for 30minutes. Negative controls included blank wells lacking TR-2-His andwells using naive G2 sera expected to lack anti-TR-2 antibodies.Positive samples were screened by ELISA a second time against TR-2-Histo confirm the identity of cells producing antibodies specific for TR-2.

The antibodies reactive with TR-2, identified above, were screened fortheir ability to induce apoptosis of WM-266 melanoma cells (ATCC Cat.No. CRL-1676) using an apoptosis assay. WM-266 cells were cultured in amicrotiter plate at a density of 4500 cells/well in normal culturemedium as recommended by ATCC overnight. For B cell cultures, 20 μL ofantigen-specific B cell culture supernatant or control B cell culturesupernatant was added to 180 μL of apoptosis medium mixture (cellculture medium containing 1 μg/mL cycloheximide (CHX) and 0.5% fetalcalf serum (“FCS”)). The culture media from the WM-266 cells was removedand the antibody-apoptosis medium mixture was added to the cells one rowat a time. The cells were incubated with the antibody-apoptosis mediumfor 20 hours to allow apoptosis to occur. The DNA-binding fluorescentdyes propidium iodide (Sigma) and Hoechst 33342 (Molecular Probes) wereadded to each well at a final concentration of 0.5 μg/mL and 2.5 μg/mL,respectively. Hoechst 33342 is membrane-permeable, and thus labels bothlive and dead cells; propidium iodide is not membrane-permeable, andthus labels only dead cells. After one hour at 37° C., images of eachwell were captured and analyzed for total number of cells (by assessingthe amount of Hoechst label) and total number of dead cells (byassessing the amount of propidium iodide label). The percent apoptosiswas determined as (propidium iodide-positive cells/Hoechst-positivecells)×100.

For XenoMax technology, the antibodies from several wells that displayedthe best induction of apoptosis were selected for rescue using thehaemolytic plaque assay. TR-2-His was biotinylated and coated ontostreptavidin-coated sheep red blood cells. Plasma cells corresponding toantigen-specific wells were thawed and incubated with the antigen-coatedred blood cells in the presence of complement and guinea pig anti-humanIgG enhancing serum. Plasma cells producing antibodies against TR-2-Hiscaused the sheep red blood cells around them to lyse and thus allowedthe identification of antigen-specific plasma cells in the mixture.Those plasma cells were isolated by micromanipulation of single cellsfrom the mixture.

After isolation of the desired single plasma cells, mRNA was extractedfrom those cells. The mRNAs encoding the heavy and light chain variablesequences were converted to cDNA and amplified by reverse transcriptasePCR using degenerate antisense primers specific for the leader sequencesand the constant regions of human IgG2 and human kappa mRNA The primersequences are provided in Table 2 below:

TABLE 2 Primer Name Primer seq AS-Ck RT 5′ GTA GGT GCT GTC CT 3′ (SEQ IDNO: 97) AS-γCH1 RT 5′ TGA GTT CCA CGA CA 3′ (SEQ ID NO: 98) AS-C Lambda5′ CTT CCA AGC CAC T 3′ (SEQ ID NO: 99) RT AS-C Lambda 5′ CA (GA) GC ACTGTC A 3′ (SEQ ID NO: 100) RT AS-Ck outer 5′ GTA GGT GCT GTC CTT GCT 3′(SEQ ID NO: 101) AS-Ck 5′ CTC TGT GAC ACT CTC CTG GGA 3′ (SEQ ID NO:102) middle AS-Ck inner 5′ GCT CTA GAT TGG AGG GCG TTA TCC ACC TTC CAC T3′ (SEQ ID NO: 103) with Xba I AS-Ck inner 5′ AAC TAG CTA GCA GTT CCAGAT TTC AAC TGC TCA TCA GAT 3′ (SEQ ID NO: 104) with Nhe I AS-CL outer5′ GCT CCC GGG TAG AAG TCA 3′ (SEQ ID NO: 105) AS-CL middle 5′ AC(CT)AGT GTG GCC TTG TTG GCT T 3′ (SEQ ID NO: 106) AS-CL inner 5′ GCT CTA GAGGG(CT) GGG AAC AGA GTG AC 3′ (SEQ ID NO: 107) ASγ-CH1 outer 5′ ACG ACACCG TCA CCG GTT 3′ (SEQ ID NO: 108) ASγ-CH1 middle 5′ AAG TAG TCC TTGACC AGG CAG CCC A 3′ (SEQ ID NO: 109) ASγ-CH1 inner 5′ GCT CTA GAG GGTGCC AGG GGG AAG ACC GAT 3′ (SEQ ID NO: 110) with Xba I (G1 specific)ASγ-CH1 inner 5′ GCT CTA GAG CAG GGC GCC AGG GGG AAG A 3′ (SEQ ID NO:111) with Xba I (G2, G3 & G4 specific) S-Vk1&2 Leader 5′ ATG AGG (CG)TCCC(CT) GCT CAG CT 3′ (SEQ ID NO: 112) outer S-Vk3 Leader 5′ ATG GAA(AG)CC CCA GC(GT) CAG CTT 3′ (SEQ ID NO: 113) outer S-Vk4 Leader 5′ ATGGTG TTG CAG ACC CAG GTC T 3′ (SEQ ID NO: 114) outer S-Vk1&2 Leader5′ GAA GAT CTC ACC ATG AGG (CG)TC CC(CT) GCT CAG CT(CT) CT 3′ (SEQ IDNO: 115) inner with BgI II S-Vk3 Leader 5′ GAA GAT CTC ACC ATG GAA(AG)CC CCA GC(GT) CAG CTT CTC TT 3′ (SEQ ID NO: 116) inner with BgI IIS-Vk4 Leader 5′ GAA GAT CTC ACC ATG GTG TTG CAG ACC CAG GTC TTC AT 3′(SEQ ID NO: 117) inner with BgI II S-VL1-4 Leader 5′ C(GA)T C(AT)C CACCAT G(GA)C (CA)(TA)G 3′ (SEQ ID NO: 118) outer S-VL1 Leader 5′ CAC CATG(GA)C C(TA)G (GC)T(CT) CCC T 3′ (SEQ ID NO: 119) outer S-VL2 Leader5′ ACC ATG GCC TGG (GA)CT C(TC)(GT) CT 3′ (SEQ ID NO: 120) outer S-VL3Leader 5′ CAC CAT GGC (CA)TG G(GA)(TC) C(CGA)(CT) T 3′ (SEQ ID NO: 121)outer S-VL4 Leader 5′ CAC CAT GGC (CT)TG G(GA)(TC) CC(CA) A(CT)T 3′ (SEQID NO: 122) outer S-VL1 Leader 5′ GAA GAT CTC ACC ATG (GA)CC (TA)G(GC)T(CT)C CCT CT 3′ (SEQ ID NO: 123) inner with BgI II S-VL2 Leader 5′ GAAGAT CTC ACC ATG GCC TGG (GA)CT C(TC) (GT) CT(CG) (TC)T 3′ (SEQ ID NO:124) inner with BgI II (Also amplifies VL5-7, 9, 10) S-VL3 Leader 5′ GAAGAT CTC ACC ATG GC(CA) TGG (GA)(TC)C (CGA)(CT)T CTC 3′ (SEQ ID NO: 125)inner with BgI II S-VL4 Leader 5′ GAA GAT CTC ACC ATG GC(CT) TGG(GA)(TC)C C(CA)A (CT)TC 3′ (SEQ ID NO: 126) inner with BgI II S-VH1Leader 5′ CAC CAT GGA (GC)TG GAC CTG GAG (GCA)(AGTC)T C 3′ (SEQ ID NO:127) outer S-VH2 Leader 5′ CAC CAT GGA CAT ACT TTG (CT)TC CAC GCT C 3′(SEQ ID NO: 128) outer S-VH3 Leader 5′ CAC CAT GGA [AG]TT [TG]GG [AG]CT[GCT][ACT]G CT 3′ (SEQ ID NO: 129) outer S-VH4 Leader 5′ CAC CAT GAA[AG]CA [TC]CT GTG GTT CTT CCT [TC]CT 3′ (SEQ ID NO: 130) outer S-VH5Leader 5′ CAC CAT GGG GTC AAC CG[CT] CAT CCT 3′ (SEQ ID NO: 131) outerS-VH6 Leader 5′ CAC CAT GTC TGT CTC CTT CCT CAT CTT C 3′ (SEQ ID NO:132) outer S-VH1 Leader 5′ GAA GAT CTC ACC ATG GA[GC] TGG ACC TGGAG[GCA] [AGTC]TC C 3′ (SEQ ID NO: 133) inner with BgI II S-VH2 Leader5′ GAA GAT CTC ACC ATG GAC ATA CTT TG[CT] TCC ACG CTC C 3′ (SEQ ID NO:134) inner with BgI II S-VH3 Leader 5′ GAA GAT CTC ACC ATGGA[AG] TT[TG] GG[AG] CT[GCT] [ACT]GC TGG (GAC)TT (SEQ ID NO: 135) innerwith TT(TC) CT 3′ BgI II S-VH4 Leader 5′ GAA GAT CT C ACC ATGAA[AG] CA[TC] CTG TGG TTC TTC CT[TC] CTC 3′ (SEQ ID NO: 136) inner withBgI II S-VH5 Leader 5′ GAA GAT CTC ACC ATG GGG TCA ACC G[CT]C ATC CT 3′(SEQ ID NO: 137) inner with BgI II S-VH6 Leader 5′ GAA GAT CTC ACC ATGTCT GTC TCC TTC CTC ATC TTC T 3′ (SEQ ID NO: 138) inner with BgI IIS-VH7 Leader 5′ GAA GAT CTC ACC ATG GAC TGG ACC TGG AGG ATC CTC TTC TTGGT 3′ (SEQ ID NO: 139) inner with BgI II

The primers introduced the following restriction sites at the 5′ end(BgIII) and the 3′ end (Xba1) of the heavy chain cDNA. Similarly, theprimers introduced the following restriction sites at the 5′ end (BgIII)and the 3′ end (NheI) of the kappa chain cDNA.

The variable heavy chain cDNA amplicon was digested with appropriaterestriction enzymes for the restriction enzyme sites that were addedduring the PCR reaction. The products of that digestion were cloned intoeach of an IgG1, IgG2, and IgG4 expression vector with compatibleoverhangs for cloning. The IgG2 and IgG4 expression vectors weredigested with BamHI and XbaI to generate compatible overhangs forsub-cloning. The IgG1 expression construct was digested with BamHI andNheI to generate compatible overhangs for sub-cloning. These vectorswere generated by cloning the constant domain of human IgG1, IgG2 orIgG4 into the multiple cloning site of the vector pcDNA3.1+/Hygro(Invitrogen).

The variable light chain cDNA amplicon was also digested withappropriate restriction enzymes for the restriction enzyme sites thatwere added during the PCR reaction. The products of that digestion werecloned into an IgK expression vector that had been digested with BamHIand NheI to provide compatible overhangs for sub-cloning. That vectorwas generated by cloning the constant domain of the human IgK gene intothe multiple cloning site of the vector pcDNA4.1+/Neo (Invitrogen).

The heavy chain and the light chain expression vectors were thenco-lipofected into a 60 mm dish of 70% confluent human embryonal kidney293 cells (ATCC, Cat. No. CRL-1573). For 24 hours, the transfected cellswere allowed to secrete a recombinant antibody with the identicalspecificity as the original plasma cell. The supernatant (3 mL) washarvested from the HEK 293 cells and the secretion of an intact antibodywas demonstrated with a sandwich ELISA to specifically detect human IgG.Control plates were coated with 2 mg/mL Goat anti-human IgG H+L O/N asfor binding plates. The plates were washed five times with water.Recombinant antibodies were titrated 1:2 for 7 wells from the undilutedlipofection supernatant. The plates were washed five times with dH2O. Agoat anti-human IgG Fc-specific HRP-conjugated antibody was added at afinal concentration of 1 μg/mL for 1 hour at room temperature for thesecretion and the two binding assays. The plates were washed five timeswith dH2O. The plates were developed with the addition oftetramethylbenzidine (TMB) for 30 minutes and the ELISA was stopped bythe addition of 1 M phosphoric acid.

In addition to the XenoMax methodology described above, certainantibodies were obtained using hybridoma technology. Immunized mice weresacrificed by cervical dislocation, and the draining lymph nodes wereharvested and pooled from each cohort. The lymphoid cells weredissociated by grinding in Dulbecco's Modified Eagle's Medium (“DMEM”)to release the cells from the tissues. Recovered cells were suspended inDMEM. The cells were counted, and 0.9 mL DMEM per 100 millionlymphocytes was added to the cell pellet to resuspend the cells gentlybut completely. The resuspended cells were incubated with 100 μL ofCD90⁺ magnetic beads per 100 million cells at 4° C. for 15 minutes. Themagnetically-labeled cell suspension (containing up to 108 positivecells (or up to 2×10⁹ total cells)) was loaded onto an LS⁺ column. Thecolumn was washed with DMEM. The total effluent was collected as theCD90⁻ negative fraction, which was expected to contain mostly B cells.

The fusion was performed by mixing washed enriched B cells from aboveand nonsecretory myeloma P3X63Ag*.653 cells (ATCC (CRL 1580, see, e.g.,Kearney et al., J. Immunol. 123, 1979, 1548-1550)) at a ratio of 1:1.The cell mixture was gently pelleted by centrifugation at 800×g. Aftercomplete removal of the supernatant from the cells, the cells weretreated with 2 to 4 ml of Pronase solution (CalBiochem; 0.5 mg/ml inphosphate-buffered saline (“PBS”)) for no more than 2 minutes. Three tofive mL of fetal bovine serum (“FBS”) was added to stop the enzymeactivity and the suspension was adjusted to a 40 mL total volume usingelectro cell fusion solution (“ECFS”) (0.3M sucrose, 0.1 mM magnesiumacetate, 0.1 mM calcium acetate). The supernatant was removed aftercentrifugation and the cells were resuspended in 40 mL ECFS. This washstep was repeated and the cells again were resuspended in 40 mL ECFS toa concentration of 2×10⁶ cells/mL.

Electro-cell fusion was performed using a fusion generator (modelECM2001, Genetronic, Inc.). The fusion chamber size used was 2.0 mL,using the following instrument settings: alignment condition: 50 V, 50seconds; membrane breaking at 3000 V, 30 microseconds; post-fusionholding time: 3 seconds.

After electro-cell fusion took place, the cell suspensions werecarefully removed from the fusion chamber under sterile conditions andtransferred into a sterile tube containing the same volume of hybridomaculture medium, containing DMEM, (JRH Biosciences), 15% FBS (Hyclone),supplemented with L-glutamine, penicillin/streptomycin, OPI(oxaloacetate, pyruvate, bovine insulin), and IL-6 (BoehringerMannheim). The cells were incubated for 15 to 30 minutes at 37° C., andthen centrifuged at 400×g (1000 rpm) for 5 minutes. The cells weregently resuspended in a small volume of hybridoma selection medium(hybridoma culture medium supplemented with 0.5× hyaluronic acid(Sigma)). The total volume was adjusted appropriately with morehybridoma selection medium based on a final plating volume of 5×10⁶ Bcells total per 96-well plate and 200 μL per well. The cells were mixedgently, pipetted into 96-well plates, and allowed to grow. On day 7 or10, one-half the medium was removed, and the cells were re-fed withfresh hybridoma selection medium.

After 14 days of culture, hybridoma supernatants were screened forTR2-specific monoclonal antibodies by ELISA. In the Primary screen, theELISA plates (Fisher, Cat. No. 12-565-136) were coated with 50 μL/wellof TR2 protein (2 μg/mL) in Coating Buffer (0.1 M Carbonate Buffer, pH9.6, NaHCO₃ 8.4 g/L), then incubated at 4° C. overnight. Afterincubation, the plates were washed with Washing Buffer (0.05% Tween 20in PBS) one time. 200 μL/well Blocking Buffer (0.5% BSA, 0.1% Tween 20,0.01% Thimerosal in 1×PBS) were added and the plates were incubated atroom temperature for 1 hour. After incubation, the plates were washedwith Washing Buffer one time. Aliquots (50 μL/well) of hybridomasupernatantsand positive and negative controls were added, and theplates were incubated at room temperature for 2 hours. The positivecontrol used throughout was serum from a hyperimmune XenoMouse animaland the negative control was serum from the KLH-immunized XenoMouseanimal. After incubation, the plates were washed three times withWashing Buffer. 100 μL/well of detection antibody goat anti-hulgGFc-HRP(Caltag Inc., Cat. No. H10507, using concentration was 1:2000 dilution)was added and the plates were incubated at room temperature for 1 hour.After incubation, the plates were washed three times with WashingBuffer. 100 μl/well of TMB (BioFX Lab. Cat. No. TMSK-0100-01) was added,and the plates were allowed to develop for about 10 minutes (untilnegative control wells barely started to show color). 50 μl/well stopsolution (TMB Stop Solution (BioFX Lab. Cat. No. STPR-0100-01) was thenadded and the plates were read on an ELISA plate reader at a wavelengthof 450 nm.

The antibodies produced by the hybridomas were analyzed using the sameapoptosis assay described above. WM-266 cells were cultured at a densityof 4500 cells/well in normal culture medium overnight in a microtiterplate. A 2× apoptosis medium mixture was prepared using cell culturemedium without FCS and additionally including 1.8 μg/mL cycloheximideand 0.9% FCS. Separate microtiter plates were used to titrate hybridomasupernatant 1:2 (in the 2× apoptosis medium mixture) in parallel with anisotype-matched negative control anti-KLH antibody. The culture mediawas removed from the WM-266 cells and 100 μL of the antibody-apoptosismedium mixture was added to each cell-containing well, one row at atime. The microtiter plates were incubated for 20 hours to allowapoptosis to occur. The DNA-binding fluorescent dyes propidium iodide(Sigma) and Hoechst 33342 (Molecular Probes) were added to each well ata final concentration of 0.5 μg/mL and 2.5 μg/mL respectively. After 1hour at 37° C., fluorescent images of each well were captured andanalyzed for total number of dead cells (PI) and total number of cells(Hoechst). The percent apoptosis was determined as (PI-positivecells/Hoechst-positive cells)×100.

Seventeen different anti-TR-2 antibodies were obtained (Antibodies A-Q)using either the XenoMax or hybridoma methodologies. All of theantibodies were sequenced, and the sequences of the heavy and lightchain variable regions identified (see FIGS. 3-19). Alignments of theheavy chains and the light chains of the seventeen antibodies are shownin FIGS. 20 and 21.

Certain antibodies were examined for their ability to induce apoptosisin cells, using a similar apoptosis assay to the one described above.WM-266 melanoma cells were cultured in a microtiter plate at a densityof 4500 cells/well in normal culture medium overnight. In a separatemicrotiter plate, the recombinant antibodies to be tested, anappropriate positive control (M413, a mouse IgG1 anti-TR-2 antibodyhaving a heavy chain variable sequence:MEVQLVESGGGLVQPGGSLKLSCAASGFTFSTYGMSWVRQTPDKRLELVALINSQGGSTYNSDSVKGRFTISRDNARNTLYLQMSSLKSEDTAMYYCARRD YESLDSWGQGTSVTVSSG(SEQ ID NO: 141) and a light chain variable sequence:DIVLTQSPASLPVSLGQRATISCRASESVEYSGTSLIQWYRQKPGQPPKLLIYAASNVDSEVPARFSGSGSGTDFSLYIHPVEEDDIAMYFCQQSRKVPWTFGG GTKLEIKRTDAAPGLEAA(SEQ ID NO: 142)), and isotype-matched negative control antibodies (apotential anti-TR2 antibody that failed to show activity) were titratedsuch that the final concentration of antibody would cover a range of0.0001 μg/mL to 5 μg/mL. The antibodies were mixed in apoptosis mediumcontaining a final concentration of 0.9 μg/mL CHX and 0.45% FCS. Theculture media was removed from the WM-266 cells and theantibody-apoptosis medium mixture was added to the cells. After 20 hoursof culture, the cells were stained with propidium iodide (Sigma) andHoechst 33342 (Molecular Probes). After 1 hour at 37° C., an image ofeach well was captured and analyzed for total number of dead cells (PI)and total number of cells (Hoechst). The percent apoptosis wasdetermined as (PI-positive cells/Hoechst-positive cells)×100.Significant cell death was observed in cells treated with M413 or withcertain anti-TR-2 antibodies described above.

Example 2 Kinetic Analyses of Anti-TR-2 Antibody Binding to TR-2

The kinetics of the binding of anti-TR-2 antibodies A to Q to TR-2 wasanalyzed using a Biacore® 2000 instrument. High-density goat anti-humanantibody surfaces were prepared on CM-5 Biacore® chips using routineamine coupling. Each purified anti-TR-2 antibody was diluted toapproximately 1 μg/ml in HBS-P running buffer containing 100 μg/ml BSA.Each anti-TR-2 antibody was captured on a separate surface using a twominute contact time and a five minute wash to stabilize the anti-TR-2antibody surface on the chip.

To analyze the kinetics of TR-2 binding to each individual anti-TR-2antibody, 226 nM recombinant human TR-2-His (described in Example 1) waskinetically injected over each anti-TR-2 surface for one minute (usingkinject) at 25° C., followed by a five minute dissociation period. Thebaseline drift resulting from a buffer injection lacking TR-2 over theanti-TR-2 antibody surface was subtracted from the observed binding oneach of the other surfaces. Additionally, the data for TR-2 binding toanti-TR-2 antibody were normalized for the amount of monoclonal antibodycaptured on each surface. Each data set was fit globally to a 1:1interaction model to determine binding kinetics. The k_(a), k_(d), andK_(d) values obtained for each antibody are shown in Table 3.

TABLE 3 Kinetics of TR-2 binding to anti-TR-2 antibody at 25° C.Antibody k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(d) (nM) A 5.3 × 10⁵ 3.7 × 10⁻³  6.9 B 5.7 × 10⁵ 1.1 × 10⁻² 19 C 6.8 × 10⁵ 2.6 × 10⁻³   3.9 D 6.2 × 10⁵2.7 × 10⁻³   4.5 E 8.7 × 10⁵ 1.8 × 10⁻³   2.1 F 3.8 × 10⁵ 5.0 × 10⁻³ 13G 6.0 × 10⁵ 1.9 × 10⁻² 31 H 8.6 × 10⁵ 8.4 × 10⁻³   9.8 I 2.9 × 10⁵ 1.3 ×10⁻³   4.4 J 5.7 × 10⁵ 7.1 × 10⁻³ 12 K 6.8 × 10⁵ 1.2 × 10⁻² 18 L 6.0 ×10⁵ 1.1 × 10⁻² 18 M 3.4 × 10⁵ 1.2 × 10⁻² 37 N 8.1 × 10⁵ 5.5 × 10⁻²  68*O 4.4 × 10⁵ 8.4 × 10⁻³ 19 P 8.1 × 10⁵ 2.7 × 10⁻²  33* Q 1.2 × 10⁶ 1.6 ×10⁻²  13* *Data for that sample exhibited heterogeneity and fit poorlyto a 1:1 model.

Example 3 Cell Killing Assays

Cell killing assays were performed with certain human anti-TR-2antibodies described in Example 2 to determine the degree to which eachantibody triggered apoptosis and cell death. Certain human anti-TR-2antibodies, as well as mouse anti-TR-2 antibodies M412 and M413, wereimmobilized in separate wells of 96-well Protein G-coated plates(reactin-bind Protein G coated plates, Pierce Cat. No. 15131). M412 is amouse IgG1 anti-TR-2 antibody having a heavy chain variable sequence:KVQLQQSGTELVKPGASVKLSCKASGYTFTEYIIHWVKQRSGQGLEWIGWFYPGSGYIKYNEKFKDKATMTADKSSSTVYMELSRLTSEDSAVYFCTRHEED GYYAAYWGQGTLVTVSA(SEQ ID NO: 143) and a light chain variable sequence:DIVMTQSHKFMSTSVGDRVSITCKASQDVSSAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDYTLTISSVQAEDLALYYCQQHYSTPYTFGGGT KLEIKR (SEQ ID NO:144). M413 is a mouse IgG1 anti-TR-2 antibody as described above inExample 1. Each antibody was added at a concentration of 50 μg/ml to afirst well, and serially diluted 1:3× in each of seven additional wells.Each antibody dilution was performed in triplicate. Plates wereincubated for 24 hours at 4° C. prior to use. Following the washing ofeach well with culture media (RPMI plus 10% FBS), one of four differentcell lines was plated onto each immobilized antibody, at a density of50,000 cells per well in a total volume of 200 μL. The cell lines testedwere COLO 205 cells (human colon adenocarcinoma), MDA-231 cells (humanbreast cancer), WM35 cells (human melanoma), and WM793 cells (humanmelanoma). Cells were incubated at 37° C./6% CO₂ for 24 hours, followedby a 6 hour incubation with ³H-thymidine. The percentage of viable cellswas assessed by determining the level of ³H-thymidine incorporation inthe treated cells relative to the level of ³H-thymidine incorporationinto the untreated cells. The ED₅₀ of each antibody was derived from thecell viability titration curve by determining the concentration ofantibody that reduced the viability of treated cells by 50% relative tountreated cells. The ED₅₀ of the human antibodies for COLO 205 cellsranged from 0 μg/ml to 3.25 μg/ml. The mouse antibodies M412 and M413had ED₅₀s of 1.85 μg/ml and 0.07 μg/ml, respectively, for those cells.The ED₅₀ of the human antibodies for MDA-231 cells ranged from 0.05μg/ml to 0.5 μg/ml. The mouse antibodies M412 and M413 had ED₅₀s of 0.6μg/ml and 0.07 μg/ml, respectively, for those cells. The ED₅₀ of thehuman antibodies for WM35 cells ranged from 0.1 μg/ml to 0.6 μg/ml. Themouse antibodies M412 and M413 had ED₅₀s of 1.85 μg/ml and 0.07 μg/ml,respectively, for those cells. The ED₅₀ of the human antibodies forWM793 cells ranged from 0.02 μg/ml to 0.2 μg/ml. The mouse antibodiesM412 and M413 had ED₅₀s of 1.85 μg/ml and 0.05 μg/ml, respectively, forthose cells.

Example 4 Human TR-2 Expression in Tumor Cell Lines

Human tumor cell lines were screened for expression of TR-2. Cell linesused included those from breast, central nervous system, colon, liver,lung, cervix, uterine, ovarian, pancreatic, prostate, and renal cancers,as well as leukemia and melanoma.

The expression of TR-2 on human tumor cells was determined using acell-based array. Briefly, 4×10⁵ cells in 100 MI CBA buffer (PBS, 3%FBS, 0.02% Azide) were distributed into each of the wells of 20 V-bottom96-well plates. CBA buffer (150 μL) was added to each well and theplates were centrifuged to spin down the cells. The medium was discardedand 100 μL of antibody solution (one of the antibodies A to Q) at 10μg/ml was added to the cell pellet resuspended in PBS containing 2% PBS(“assay buffer”). After a 25 minute incubation on ice, the cells werewashed once in assay buffer. 100 μL of a secondary goat anti-human IgGFc-specific horseradish peroxidase (HRP, Pierce) was added to the wells,and the plates were incubated on ice for 20 minutes. The plates werewashed twice with assay buffer, and 100 μL of the TMB substrate (ZYMED)was added for 10 minutes at room temperature. The plates werecentrifuged and 50 μL of each supernate was transferred into a cleanplate containing 50 μL stop solution (BioFX Laboratories). Opticaldensity readings were performed at 450 nm using the SpectraMax/plusreader (Molecular Devices). The data were normalized by subtracting theoptical density values obtained from an isotype control antibody.

Several cell lines had an OD₄₅₀ greater than 0.1 in the assay, includingbreast cancer cell lines HS 578.T (OD of 0.122) and T-47D (OD of 0.112),colon cancer cell lines TE 671(u) (OD of 0.109), HT-29 (OD of 0.193),SW-948 (OD of 0.122), KM-12 (OD of 0.354), and HCC-2998 (OD of 0.133),liver cancer cell lines NCI-N87 (OD of 0.154) and NCI-SNU-5 (OD of0.137), leukemia cell lines HL-60 (OD of 0.233) and hPBMC (OD of 0.131),non-small-cell lung cancer cell line JY (OD of 0.118), CCRF-CEM (OD of0.106), NCI-H2126 (OD of 0.108) and NCI-H460 (OD of 0.122), melanomacell lines SK-mel-5 (OD of 0.131), LOX IMVI (OD of 0.102), RPMI 7951 (ODof 0.101), and UACC-62 (OD of 0.127), pancreas cancer cell lines HPAF II(OD of 0.117) and CAPAN-1 (OD of 0.101), prostate cancer cell line LNCaP(OD of 0.174), and renal carcinoma cell lines Caki-1 (OD of 0.148) andUO-31 (OD of 0.104). The greatest expression of TR-2 among the tumorcell lines studied was found in colon cancer cell lines KM-12 and HT-29,and in leukemia cell line HL-60. None of the central nervous system,small-cell liver, cervical, uterine, or ovarian cancer cell linesstudied had an OD450 greater than background.

To determine TR-2 expression profile on human tumor cell lines, theabove human tumor cell lines were assayed with the mouse anti-TR-2antibody M412. The expression of TR-2 on human tumor cells wasdetermined using a cell-based array. Briefly, 4×10⁵ cells in 100 MI CBAbuffer (PBS, 3% FBS, 0.02% Azide) were distributed into each of thewells of 20 V-bottom 96-well plates. CBA buffer (150 μL) was added toeach well and the plates were centrifuged to spin down the cells. Themedium was discarded and 100 μL of mouse anti-TR-2 monoclonal antibodyM412 at 10 μg/ml was added to the cell pellet resuspended in PBScontaining 2% PBS (“assay buffer”). After a 25 minute incubation on ice,the cells were washed once in assay buffer. 100 μL of a secondary goatanti-mouse IgG Fc-specific horseradish peroxidase (HRP, Pierce) wasadded to the wells, and the plates were incubated on ice for 20 minutes.The plates were washed twice with assay buffer, and 100 μL of the TMBsubstrate (ZYMED) was added for 10 minutes at room temperature. Theplates were centrifuged and 50 μL of each supernate was transferred intoa clean plate containing 50 μL stop solution (BioFX Laboratories).Optical density readings were performed at 450 nm using theSpectraMax/plus reader (Molecular Devices). The data were normalized bysubtracting the optical density values obtained from an isotype controlantibody.

Many of the cell lines had TR-2 expression. The highest expressors(those with an OD450 nm greater than 0.3) included breast cancer celllines HS 578.T (OD of 0.403), MDA-MB-231 (OD of 0.408), and T-47D (OD of0.366), CNS cancer cell lines SF-295 (OD of 0.354) and U251 (OD of0.323), colon cancer cell lines HCT-116 (OD of 0.41), HT-29 (OD of0.869), SW-707 (OD of 0.323), SW-948 (OD of 0.423), KM-12 (OD of 0.77),and HCC-2998 (OD of 0.635), liver cancer cell line NCI-SNU-1 (OD of0.354), leukemia cell line A 673 (OD of 0.347), non-small-cell lungcancer cell lines HOP-62 (OD of 0.313), HOP-62 (OD of 0.47), NCI-H2126(OD of 0.501), NCI-H460 (OD of 0.326), small cell lung cancer line A549(OD of 0.381), melanoma cell lines LOX IMVI (OD of 0.573), RPMI 7951 (ODof 0.322), and UACC-62 (OD of 0.319), ovarian cancer cell line IGROV1(OD of 0.312), prostate cancer cell lines DU 145 (OD of 0.372), 22Rv1(OD of 0.301), and LNCaP (OD of 0.63), and renal carcinoma cell linesCaki-1 (OD of 0.93), Caki-2 (OD of 0.443), SN12C (OD of 0.313), andUO-31 (OD of 0.331). The greatest expression of TR-2 among the tumorcell lines treated with mouse anti-TR-2 antibody was found in renalcarcinoma cell line Caki-1, and in colon cancer cell lines HT-29 andKM-12.

Example 5 Antibody Cross-Reactivity

The ability of certain of the human anti-TR-2 antibodies to block thebinding of the others to TR-2 was assessed, as described in Jia et al.,J. Immunol. Methods 288: 91-98 (2004). The beads were conjugated withanti-human IgG antibodies using the coupling procedure taken directlyfrom the Luminex 100 User's Manual, Version 1.7. After the beads wereactivated, they were coupled to a Pharmingen mouse anti-hIgG mAb,following the manufacturer's instructions. Two experiments wereperformed. In a first experiment, the coated beads were incubated fortwo hours at room temperature. In a second experiment, the coated beadswere incubated overnight at 4° C. At the end of the incubation, thecoated beads were blocked and then counted using a Coulter cell counter.Conjugated beads were either used immediately or were stored at 4° C. inthe dark for future use.

The categorization of the anti-TR-2 antibodies based on epitopecross-reactivity was performed by the following steps. First, each setof bead-mouse anti-hIgG complexes from above were separately incubatedwith a reference antibody (“reference antibody”) on a rotator overnightat 4° C. The reference antibody was selected from anti-TR-2 antibodiesA-Q, described above. After antibody capture, 2000 of each bead-mouseanti-hIgG-reference Ab complexes were pooled together in one tube, andthen immediately added to each well of a 96-well plate and aspirated.TR-2 (50 ng) was added to each well and incubated for 1 hour at roomtemperature. After washing the wells, 100-500 ng/mL of another of thehuman anti-TR-2 antibodies (the “probe antibody”) was added to each welland incubated for 2 hours at room temperature. After washing the wells,bound probe antibody was detected using 1 μg/ml of a biotinylatedversion of the same monoclonal mouse anti-hIgG used for capturing thereference antibody. Following incubation and washing of the wells, 0.5μg/ml streptavidin-phycoerythrin was added. The mixture was incubatedfor 30 minutes at room temperature and then the phycoerythrin signal wasdetected using the Luminex 100. An additional set of wells lackingantigen was used as a negative control to aid in data analysis.

The data was analyzed in a two-step process. First, the data wasnormalized using the negative control values. Second, the anti-TR-2antibodies were clustered according to their ability to impede bindingof one or more other anti-TR-2 antibodies. For the clustering analysis,a dissimilarity matrix was generated from the normalized intensitymatrix. Antibodies were clustered based on the values in the averagedissimilarity matrix using the SPLUS 2000 agglomerative nestinghierarchical clustering subroutine with the Manhattan metric, using aninput dissimilarity matrix of the actual average dissimilarity matrix.

Based on the findings, the antibodies were placed into four differentepitope groups. Within any one group, the binding of one of the groupmembers to TR-2 blocks the binding of another member of the same groupto TR-2. However, the binding of one of the members of group 1 to TR-2,for example, does not block the binding of one of the members of groups2, 3, or 4 to TR-2. Those groups are shown in FIG. 22.

Example 6 Epitope Mapping

To identify the specific region of TR-2 important for binding to certaindescribed anti-TR-2 antibodies, an epitope mapping study was performed.An N-avidin-TR-2 construct was made by PCR-amplifying the codingsequence for mature TR-2 (MacFarlane, 1997) from a template source andcloning it into a pCEP4 vector (Invitrogen) containing the chickenavidin sequence in an orientation such that upon insertion at a HindIIIsite, the TR-2 sequence was joined at the C-terminus of the avidinsequence. The forward primer for the mature TR-2 coding sequence wasGTAAGCAAGCTTGGCTC TGATCACCCAACAGA (SEQ ID NO: 145), and the reverseprimer was GATTAGGGATCCAGAGGCAGGAGTCCCTGG (SEQ ID NO: 146). The aminoacid sequence of the resulting avidin-TR-2 fusion protein wasMVHATSPLLLLLL LSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLALITQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKESGTKHSGEAPAVEETVTSSPGTPAS (SEQ ID NO: 69).

Twelve molecules comprising N-avidin and truncations of human TR-2 weresynthesized as described below. Three molecules had only C-terminaltruncations of human TR-2 (TR-2-1 through TR-2-3), and nine moleculeshad truncations at both the N- and the C-terminus of human TR-2 (TR-2-4through TR-2-13) (shown schematically in FIG. 23). Polynucleotidesencoding human TR-2 truncations were prepared by PCR amplification usingthe primers described below. To form each of the twelve molecules, thetruncated human TR-2 resulting from the amplification was inserted intothe pCEP4 vector (Invitrogen) containing the chicken avidin sequencethat is described above. The polynucleotide encoding amino acids 1-43 ofmature TR-2 was amplified using the forward primerGTAAGCAAGCTTGGCTCTGATCACCCAACAAGA (SEQ ID NO: 145) and the reverseprimer TAGTTGGGATCCTCAGGAGATGCAATCTCT ACCGT (SEQ ID NO: 147). The aminoacid sequence of TR-2-1 wasMVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLALITQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCIS (SEQ ID NO: 70).

The polynucleotide encoding amino acids 1-85 of mature TR-2 wasamplified using the forward primer GTAAGCAAGCTTGGCTCTGATCACCCAACAAGA(SEQ ID NO: 145) and the reverse primerGGTAGTGGATCCTCACTGACACACTGTGTTTCTGG (SEQ ID NO: 148). The amino acidsequence of TR-2-2 wasMVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLALITQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQ (SEQ ID NO: 71).

The polynucleotide encoding amino acids 1-126 of mature TR-2 wasamplified using the forward primer GTAAGCAAGCTTGGCTCTGATC ACCCAACAAGA(SEQ ID NO: 145) and the reverse primer GTAATGGGATCCTCAGACACATTCGATGTCACTCC (SEQ ID NO: 149). The amino acid sequence ofTR-2-3 was MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLALITQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECV (SEQ ID NO: 72).

The polynucleotide encoding amino acids 16-43 of mature TR-2 wasamplified using the forward primer GTAATGAAGCTTGCCACAACA AAAGAGGTCCAG(SEQ ID NO: 150) and the reverse primer TAGTTGGGATCCTCAGGAGATGCAATCTCTACCGT (SEQ ID NO: 147). The amino acid sequence ofTR-2-4 was MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLPQQKRSSPSEGLCPPGHHISEDGRDCIS (SEQ ID NO: 73).

The polynucleotide encoding amino acids 16-85 of mature TR-2 wasamplified using the forward primer GTAATGAAGCTTGCCACAACAAA AGAGGTCCAG(SEQ ID NO: 150) and the reverse primer GGTAGTGGATCCTCACTGACACACTGTGTTTCTGG (SEQ ID NO: 148). The amino acid sequence ofTR-2-5 was MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQ (SEQ ID NO: 74).

The polynucleotide encoding amino acids 16-126 of mature TR-2 wasamplified using the forward primer GTAATGAAGCTTGCCACAACAAA AGAGGTCCAG(SEQ ID NO: 150) and the reverse primer GTAATGGGATCCTCAGACACATTCGATGTCACTCC (SEQ ID NO: 149). The amino acid sequence of TR-2-6was MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCP RGMVKVGDCTPWSDIECV(SEQ ID NO: 75).

The polynucleotide encoding amino acids 42-85 of mature TR-2 wasamplified using the forward primer GATTGAAAGCTTGATCTCCTGCAAATATGGACAG(SEQ ID NO: 151) and the reverse primerGGTAGTGGATCCTCACTGACACACTGTGTTTCTGG (SEQ ID NO: 148). The amino acidsequence of TR-2-7 was MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQ (SEQ ID NO: 76).

The polynucleotide encoding amino acids 42-126 of mature TR-2 wasamplified using the forward primer GATTGAAAGCTTGATCTCCTGCAAATATGGACAG(SEQ ID NO: 151) and the reverse primerGTAATGGGATCCTCAGACACATTCGATGTCACTCC (SEQ ID NO: 149). The amino acidsequence of TR-2-9 was MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECV (SEQ ID NO: 77).

The polynucleotide encoding amino acids 85-154 of mature TR-2 wasamplified using the forward primer GTAATGAAGCTTGCAGTGCGAAGAAGGCACCT (SEQID NO: 152) and the reverse primer GATTAGGGATCCAGAGGCAGGAGTCCCTGG (SEQID NO: 146). The amino acid sequence of TR-2-10 wasMVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKESGTKHSGEAPAVEETVTSSPGTPAS (SEQ ID NO: 78).

The polynucleotide encoding amino acids 42-154 of mature TR-2 wasamplified using the forward primer GATTGAAAGCTTGATCTCCTGC AAATATGGACAG(SEQ ID NO: 151) and the reverse primer GATTAGGGATCCA GAGGCAGGAGTCCCTGG(SEQ ID NO: 146). The amino acid sequence of TR-2-11 wasMVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKESGTKHS GEAPAVEETVTSSPGTPAS(SEQ ID NO: 79).

The polynucleotide encoding amino acids 16-66 of mature TR-2 wasamplified using the forward primer TGATTGAAGCTTGCCACAACAA AAGAGGTCCAG(SEQ ID NO: 150) and the reverse primer GATGGAGGATCCTCAACACCTGGTGCAGCGCAAG (SEQ ID NO: 153). The amino acid sequence ofTR-2-12 was MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLPQQKRSSPSEGLCPPGHHISEDGRDCISYKYGQDYSTHWND LLFCLRCTRC (SEQ IDNO: 80).

The polynucleotide encoding amino acids 16-74 of mature TR-2 wasamplified using the forward primer TGATTGAAGCTTGCCACAACA AAAGAGGTCCAG(SEQ ID NO: 150) and the reverse primer GTAAGTGGATCCTCAGCAGGGACTTAGCTCCACT (SEQ ID NO: 154). The amino acid sequence ofTR-2-13 was MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHW NDLLFCLRCTRCDSGEVELS(SEQ ID NO: 81). Four molecules comprising N-avidin and truncations ofTR-2 from cynomolgus monkey were synthesized as described below. Thepolynucleotide encoding amino acids 1 to 132 of mature cyno TR-2 wasamplified using the forward primer GTTAGTAAGCTTGGCTCCAATCACCCGAC (SEQ IDNO: 155) and the reverse primer GTTGATGGATCCTTCTTTGTGGACACTCGAT (SEQ IDNO: 156). The amino acid sequence of cyno TR-2 (short) was MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLAPITRQSLDPQRRAAPQQKRSSPTEGLCPPGHHISEDSRDCISCKYGQDYSTHWNDFLFCLRCTKCDSGEVEVSSCTTTRNTVCQCEEGTFREEDSPEICRKCRTGCPRGMVKVKDCTPWSDIECPQRRIQT (SEQ ID NO: 82).

The polynucleotide encoding amino acids 1 to 154 of mature cyno TR-2 wasamplified using the forward primer GTTAGTAAGCTTGGCTCCA ATCACCCGAC (SEQID NO: 155) and the reverse primer GTAGTTGGATCCTC AAGAAGCAGGAGTCCCAGGG(SEQ ID NO: 157). The amino acid sequence of cyno TR-2 (long) wasMVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLAPITRQSLDPQRRAAPQQKRSSPTEGLCPPGHHISEDSRDCISCKYGQDYSTHWNDFLFCLRCTKCDSGEVEVSSCTTTRNTVCQCEEGTFREEDSPEICRKCRTGCPRGMVKVKDCTPWSDIECVHKESGTKHTGEV PAVEKTVTTSPGTPAS (SEQID NO: 83).

The polynucleotide encoding amino acids 1 to 85 of mature cyno TR-2 wasamplified using the forward primer GTTAGTAAGCTTGGCTCCA ATCACCCGAC (SEQID NO: 155) and the reverse primer GTATGAGGGATCCTC ACTGACACACCGTGTTTCTGG(SEQ ID NO: 158). The amino acid sequence of cyno 1-85 wasMVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLAPITRQSLDPQRRAAPQQKRSSPTEGLCPPGHHISEDSRDCISCKYGQDYSTHWNDFLFCLRCTKCDSGEVEVSSCTTTRNTVCQ (SEQ ID NO: 84).

The polynucleotide encoding amino acids 16 to 85 of mature cyno TR-2 wasamplified using the forward primer GTATGGAAGCTTGCCACAA CAAAAGAGATCCAGC(SEQ ID NO: 159) and the reverse primer GTATGAGGGATCCTCACTGACACACCGTGTTTCTGG (SEQ ID NO: 158). The amino acid sequence ofcyno 16-85 was MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLPQQKRSSPIEGLCPPGHHISEDSRDCISCKYGQDYSTHWNDFLFCLRCTKCDSGEVEVSSCTTTRNTVCQ (SEQ ID NO: 85).

Four N-avidin-fused chimeras were also made using different portions ofhuman TR-2 and cyno TR-2, as shown in FIG. 25. Each chimera wasconstructed by preparing two PCR products with overlapping ends thatwere then amplified together using the same 5′ and 3′ primers. To formeach of the chimeras, the amplified polynucleotide was then subclonedinto the pCEP4 vector (Invitrogen) containing the chicken avidinsequence that is described above. An alignment of the human, cyno(short), and mouse TR-2 sequences is shown in FIG. 26.

Cyno/human chimera #1 was prepared by amplifying a region of mature cynoTR-2 corresponding to amino acids 1-16 using the forward primerGTTAGTAAGCTTGGCTCCAATCACCCGAC (SEQ ID NO: 155) and the reverse primerGGACCTCTTTTGTTGTGGAGCCGCTCTTCGCTGG (SEQ ID NO: 159) and amplifying aregion of mature human TR-2 corresponding to amino acids 17-85 using theforward primer CAGCGAAGAGCGGCTCCACAACAAAAG AGGTCCAG (SEQ ID NO: 160) andthe reverse primer GGTAGTGGATCCTCACT GACACACTGTGTTTCTGG (SEQ ID NO:148). Overlapping PCR of the cyno and human TR-2 fragments was performedusing the forward primer for the cyno TR-2 amino acids 1-16 fragment,above (SEQ ID NO: 155) and the reverse primer for the human TR-2 aminoacids 17-85 fragment, above (SEQ ID NO: 148). The amino acid sequencefor cyno/human chimera #1 was MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLAPITRQSLDPQRRAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTV CQ (SEQ ID NO: 86).

Cyno/human chimera #2 was prepared by amplifying a region of mature cynoTR-2 corresponding to amino acids 1-16 using the forward primerGTTAGTAAGCTTGGCTCCAATCACCCGAC (SEQ ID NO: 155) and the reverse primerGGACCTCTTTTGTTGTGGAGCCGCTCTTCGCTGG (SEQ ID NO: 159) and amplifying aregion of mature human TR-2 corresponding to amino acids 17-154 usingthe forward primer CAGCGAAGAGCGGCTCCACAACAAAA GAGGTCCAG (SEQ ID NO: 160)and the reverse primer GATTAGGGATCCTCAA GAGGCAGGAGTCCCTGG (SEQ ID NO:146). Overlapping PCR of the cyno and human TR-2 fragments was performedusing the forward primer for the cyno TR-2 amino acids 1-16 fragment,above (SEQ ID NO: 155) and the reverse primer for the human TR-2 aminoacids 17-154 fragment, above (SEQ ID NO: 146). The amino acid sequencefor cyno/human chimera #2 was MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLAPITRQSLDPQRRAAPQQKRSSPSEGLCPPGHHISEDGRDYISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKESGTKHSGEAPAVEETVTSSPGTPAS (SEQ ID NO: 87).

Cyno/human chimera #3 was prepared by amplifying a region of maturehuman TR-2 corresponding to amino acids 1-16 using the forward primerGTAAGCAAGCTTGGCTCTGATCACCCAACAAGA (SEQ ID NO: 145) and the reverseprimer GGATCTCTTTTGTTGTGGGGCCGCTCTCTGCTGG G (SEQ ID NO: 161) andamplifying a region of mature cyno TR-2 corresponding to amino acids17-85 using the forward primer CAGCAGAGAGCGGCCCCACA ACAAAAGAGATCCAGC(SEQ ID NO: 162) and the reverse primer GTATGAGGGATCCTCACTGACACACCGTGTTTCTGG (SEQ ID NO: 158). Overlapping PCR of thecyno and human TR-2 fragments was performed using the forward primer forthe human TR-2 amino acids 1-16 fragment, above (SEQ ID NO: 145) and thereverse primer for the cyno TR-2 amino acids 17-85 fragment, above (SEQID NO: 158). The amino acid sequence for cyno/human chimera #3 wasMVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEiKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFiDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLALITQQDLAPQQRAAPQQKRSSPTEGLCPPGHHISEDSRDCISCKYGQDYSTHWNDFLFCLRCTKCDSGEVEVSS CTTTRNTVCQ (SEQ IDNO: 88).

Cyno/human chimera #4 was prepared by amplifying a region of maturehuman TR-2 corresponding to amino acids 1-16 using the forward primerGTAAGCAAGCTTGGCTCTGATCACCCAACAAGA (SEQ ID NO: 145) and the reverseprimer GGATCTCTTTTGTTGTGGGGCCGCTCTCTGCTGG G (SEQ ID NO: 161) andamplifying a region of mature cyno TR-2 corresponding to amino acids17-154 using the forward primer CAGCAGAGAGCGGCCCCACA ACAAAAGAGATCCAGC(SEQ ID NO: 162) and the reverse primer GTAGTTGGATCCTCAAGAAGCAGGAGTCCCAGGG (SEQ ID NO: 157). Overlapping PCR of the cynoand human TR-2 fragments was performed using the forward primer for thehuman TR-2 amino acids 1-16 fragment, above (SEQ ID NO: 145) and thereverse primer for the cyno TR-2 amino acids 17-154 fragment, above (SEQID NO: 157). The amino acid sequence for cyno/human chimera #4 wasMVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLALITQQDLAPQQRAAPQQKRSSPTEGLCPPGHHISEDSRDCISCKYGQDYSTHWNDFLFCLRCTKCDSGEVEVSSCTTTRNTVCQCEEGTFREEDSPEICRKCRTGCPRGMVKVKDCTPWSDIECVHKESGTKHTGEVPAVEKTVTTSPGTPAS (SEQ ID NO: 89). Four additional modifiedTR-2 proteins were constructed by replacing short regions of human TR-2with the corresponding mouse TR-2 sequence, in the context of anN-avidin fusion. Human/mouse TR-2 #1 comprised the mouse TR-2 sequencefrom amino acids 1-22 and the human TR-2 sequence from amino acids23-150. Human/mouse TR-2 #2 comprised the human TR-2 sequence from aminoacids 1-28 and 35-150 and the mouse TR-2 sequence from amino acids29-34. Human/mouse TR-2 #3 comprised the human TR-2 sequence from aminoacids 1-53 and 60-150 and the mouse TR-2 sequence from amino acids54-59. Human/mouse TR-2 #4 comprised the human TR-2 sequence from aminoacids 1-66 and 76-150 and the mouse TR-2 sequence from amino acids67-75. To form each of the modified proteins, the amplifiedpolynucleotide was then subcloned into the pCEP4 vector (Invitrogen)containing the chicken avidin sequence that is described above.

Human/mouse TR-2 #1 was prepared by amplifying a region of mature humanTR-2 corresponding to amino acids 23-150 using the forward primerCAGCGGCCGGAGGAGAGCCCCTCAGAGGGATTGT (SEQ ID NO: 163) and the reverseprimer GATTGAGGATCCCTAAGAGGCAGGAGTCCCTGG (SEQ ID NO: 164) and amplifyinga region of mature mouse TR-2 corresponding to amino acids 1-22 usingthe forward primer TGAATGAAGCTTGGTTCCAGTA ACAGCTAACCCA (SEQ ID NO: 165)and the reverse primer TCCCTCTGAGGG GCTCTCCTCCGGCCGCTGTAG (SEQ ID NO:166). Overlapping PCR of the human and mouse TR-2 fragments wasperformed using the forward primer for the mouse TR-2 amino acids 1-22fragment, above (SEQ ID NO: 165) and the reverse primer for the humanTR-2 amino acids 23-150 fragment, above (SEQ ID NO: 164). The amino acidsequence for human/mouse TR-2 #1 was MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLVPVTANPAHNRPAGLQRPEESPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKESGTKHSGEAPAVEETVTSSPGTPAS (SEQ ID NO: 90).

Human/mouse TR-2 #2 was prepared by amplifying a region of mature humanTR-2 corresponding to amino acids 1-28 using the forward primerGTAAGCAAGCTTGGCTCTGATCACCCAACAAGA (SEQ ID NO: 145) and the reverseprimer CAGGTACTGGCCTGCTAGACACAATCCCTCTGAGGGG (SEQ ID NO: 167),amplifying a region of mature human TR-2 corresponding to amino acids35-150 using the forward primer CTAGCAGGCCAGTACCTGTCAG AAGACGGTAGAGATTGC(SEQ ID NO: 168), and the reverse primer GATTGAGGATCCCTAAGAGGCAGGAGTCCCTGG (SEQ ID NO: 164) and amplifying a region ofmature mouse TR-2 corresponding to amino acids 29-34 using the forwardprimer CAGGTACTGGCCTGCTAGACACAATCCCTCTGAGGGG (SEQ ID NO: 169) and thereverse primer CTAGCAGGCCAGTACCTGTCAGAAGACGG TAGAGATTGC (SEQ ID NO:170). Overlapping PCR of the human and mouse TR-2 fragments wasperformed using the forward primer for the human TR-2 amino acids 1-28fragment, above (SEQ ID NO: 145) and the reverse primer for the humanTR-2 amino acids 35-150 fragment, above (SEQ ID NO: 170). The amino acidsequence for human/mouse TR-2 #2 was MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQP TFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLALITQQDLAPQQRAAPQQKRSSPSEGLCLAGQYLSEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKESGTK HSGEAPAVEETVTSSPGTPAS(SEQ ID NO: 91).

Human/mouse TR-2 #3 was prepared by amplifying a region of mature humanTR-2 corresponding to amino acids 1-53 using the forward primerGTAAGCAAGCTTGGCTCTGATCACCCAACAAGA (SEQ ID NO: 145) and the reverseprimer TGAATCCAGAGAATGGTTGGAGTGAGTGCTATAGTCCTG TC (SEQ ID NO: 171), andamplifying a region of mature human TR-2 corresponding to amino acids60-154 using the forward primer TCCAACCATTCTCTGGATTCA TGCTTGCGCTGCACCAGG(SEQ ID NO: 172) and the reverse primer GATTGAGGATCCCTAAGAGGCAGGAGTCCCTGG (SEQ ID NO: 173) The above primers includenucleotides encoding mouse TR-2 corresponding to amino acids 54-59.Overlapping PCR of the human and mouse TR-2 fragments was performedusing the forward primer for the human TR-2 amino acids 1-53 fragment,above (SEQ ID NO: 145) and the reverse primer for the human TR-2 aminoacids 60-154 fragment, above (SEQ ID NO: 173). The amino acid sequencefor human/mouse TR-2 #3 was MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLALITQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKESGT KHSGEAPAVEETVTSSPGTPAS(SEQ ID NO: 92).

Human/mouse chimera #4 was prepared by amplifying a region of maturehuman TR-2 corresponding to amino acids 1-66 using the forward primerGTAAGCAAGCTTGGCTCTGATCACCCAACAAGA (SEQ ID NO: 145) and the reverseprimer TCGGGTTTCTACGACTTTATCTTCCTTACACCTGG TGCAGCGCAAG (SEQ ID NO: 174),and amplifying a region of mature human TR-2 corresponding to aminoacids 76-154 using the forward primer AAGGAAGATAAAGTCGTAGAAACCCGATGCACCACGACCAGAAAC AC (SEQ ID NO: 175) and thereverse primer GATTGAGGATCCCTAAGAGGCA GGAGTCCCTGG (SEQ ID NO: 176). Theabove primers include nucleotides encoding mouse TR-2 corresponding toamino acids 67-75. Overlapping PCR of the human and mouse TR-2 fragmentswas performed using the forward primer for the human TR-2 amino acids1-66 fragment, above (SEQ ID NO: 145) and the reverse primer for thehuman TR-2 amino acids 76-154 fragment, above (SEQ ID NO: 176). Theamino acid sequence for human/mouse TR-2 #4 was MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLALITQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHSNHSLDSCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKESGTKHSGEAPAVEETVTSSPGTPAS (SEQ ID NO: 93).

Expression of avidin fusion proteins was performed by transienttransfection of human 293T adherent cells in vented T75 tissue cultureflasks. Cells were grown and maintained in DMEM with 10% dialyzed FBSand 1× pen-step-glutamine at 37° C. with 5% CO₂. To prepare fortransfection, approximately 3×10⁶ 293T cells were inoculated into eachof a series of clean T75 flasks containing 15 ml growth medium, and allof the flasks were grown overnight for approximately 20 hours. Each ofthe pCEP4-Avidin(N)-TR-2 constructs were transfected into differentcells as follows. 15 μg DNA was mixed with 75 μL Lipofectamine 2000(Invitrogen) in the presence of Opti-MEM medium (Invitrogen) to form aDNA-Lipofectamine complex. The complex was incubated for 20 minutes.During that incubation period, the growth medium was aspirated from theT75 flasks and replaced with 15 mL Opti-MEM. Following incubation, eachtransfection complex was inoculated into a different flask and incubatedat 37° C. for 4 to 5 hours. At the end of the incubation period, theOpti-MEM medium in each flask was replaced with fresh growth medium.Approximately 48 hours post-transfection, the conditioned media washarvested and transferred to 50 ml tubes (Falcon). The tubes werecentrifuged at 2000×g for 10 minutes at 4° C. to remove cells anddebris, and subsequently transferred to a clean 50 mL tube. A controlflask lacking transfected DNA was also made following the same protocol,yielding negative control conditioned media for binding experiments.

The concentration of each N-avidin-TR-2 fusion protein was determinedusing a quantitative FACS-based assay. The avidin fusion proteins werecaptured on 6.7 μm biotin polystyrene beads (Spherotech, Inc.). Twosamples were prepared for each fusion protein: 5 μL (approximately3.5×10⁵) bead suspension plus 20 μL of 1× conditioned media, and 5 μLbead suspension plus 200 μL of 1× conditioned media. All samples wereincubated for 1 hour at room temperature with rotation. Conditionedmedia was removed from each sample by centrifugation and washing withPBS containing 0.5% BSA (BPBS). The avidin beads were stained with 200μL of a 0.5 μg/mL solution of a goat FITC-labeled anti-avidin antibody(Vector Labs, Burlingame, Calif.) in BPBS. The reaction was allowed toproceed at room temperature for 45 minutes with the reaction tubescovered by foil. Following incubation, the beads were collected again bycentrifugation and washing with BPBS, and resuspended for analysis in0.5 ml BPBS. The FITC fluorescence was detected using a FACScan (BectonDickinson Bioscience). The signal was converted to protein mass using astandard curve derived with recombinant avidin.

The binding of two human anti-TR-2 antibodies to each of the human TR-2truncations, to human TR-2, and to TR-2 from cynomolgus monkey wasassessed. The binding assay was performed as follows. Biotin beads,described above, were loaded with approximately 100 ng of one of theN-Avidin TR-2 fusion proteins per 3.5×10⁵ beads and brought to volumewith growth medium. The beads were mixed with 1 μg of FITC-conjugatedhuman anti-TR-2 monoclonal antibody in 0.2 mL BPBS. After incubation for1 hour at room temperature, 3 mL BPBS was added and the antibody-beadcomplexes were collected by centrifugation for 5 minutes at 750×g. Thepellet was washed in 3 mL BPBS. The antibody bound to the avidin-beadcomplexes was detected by FACS analysis. The mean fluorescent intensitywas recorded for each sample. Binding of those antibodies to conditionedmedia lacking TR-2 was used as a negative control (“Neg CM”). Theresults are shown in FIG. 24.

The observed binding patterns of the two antibodies were similar. Thestrongest observed binding was to the positive control, human TR-2, withan average fluorescent intensity of 7349. Observed binding of theantibodies (as measured in fluorescent intensity) to truncation TR-2-2was 6561-6693, to truncations TR-2-3 and TR-2-5 was 3158-3866, totruncation TR-2-6 was 1959-2202, and to truncation TR-2-1 was 662-759.Binding of the antibodies to full-length TR-2 from cynomolgus monkey (asmeasured in fluorescent intensity) was 666-764. The antibodies did notbind to mouse or rat TR-2, or to truncations TR-2-4, TR-2-7, TR-2-9,TR-2-10, TR-2-11, TR-2-12, or TR-2-13, as determined by the fact thatthe binding was similar to the background for the experiment.

TR-2-1 is a C-terminal truncation of TR-2 after amino acid 43, andTR-2-2, -3, -5, and -6 all include at least amino acids 16 to 85.Binding occurred when the entire region from amino acids 1 to 85 waspresent (see results for TR-2-2). The addition of amino acids 86 to 126decreased binding by approximately two-fold (compare results for TR-2-2to TR-2-3). The absence of amino acids 1 to 15 from the N-terminus ofTR-2 in TR-2-2 decreased binding by approximately two-fold (compareresults for TR-2-2 to TR-2-5). The simultaneous absence of amino acids 1to 15 and the addition of amino acids 86 to 126 decreased binding byapproximately three-fold (compare results for TR-2-2 to TR-2-6).Elimination of residues 44 to 85 (TR-2-1) reduced binding to about 11%of that observed to TR-2-2. Those results indicate that one or moreresidues in the regions of amino acids 1 to 15 (SEQ ID NO: 94;ALITQQDLAPQQRAA) and 44 to 85 (SEQ ID NO: 95; CKYGQDYSTHWNDLLFCLRCTRCDSGEVE LSPCTTTRNTVCQ) are important for binding of those twohuman anti-TR-2 antibodies and human TR-2.

The binding of a human anti-TR-2 antibody to each of the cyno TR-2truncations, to human/cyno chimeras, and to human TR-2 comprisingcertain mouse TR-2 domains was also assessed. The anti-TR-2 antibodybound strongly to full-length human TR-2 (fluorescent intensity (“FI”)of 5681). The binding of the anti-TR-2 antibody to the full-length longversion of cyno TR-2 was about five-fold reduced (FI of 1573) from thatto full-length human TR-2. Only background levels of binding wereobserved to the full-length short version of cyno TR-2 (FI of 209) andto cyno TR-2 truncations 17-154 (FI of 51), cyno 1-85 (FI of 11), andcyno 17-85 (FI of 8).

The binding of certain human anti-TR-2 antibodies to cyno/human TR-2chimeras was also assessed (see FIG. 27). Observed binding (FI) of theantibodies to the four chimeras was as follows: cyno/human chimera #1:FI of 5977; cyno/human chimera #2: FI of 47; cyno/human chimera #3: FIof 12; cyno/human chimera #4: FI of 1507. As above, observed binding ofthe antibodies to full-length human TR-2 was 5681, while binding of theantibodies to full-length cyno TR-2 was 1573 (long form) and 209 (shortform).

Because the antibody binding to cyno/human chimera #1 was similar tothat to the truncation TR-2-5, replacement of amino acids 1-16 with thecorresponding cyno sequence apparently did not affect antibody bindingin the context of human amino acids 17-85. However, replacement of aminoacids 1-16 with the corresponding cyno sequence in the context of thefull-length human TR-2 (cyno/human #2) significantly abrogated binding,confirming that at least one amino acid in the region from 1-16 formspart of the epitope. Binding to cyno/human chimeras #3, and #4 wassignificantly attenuated from that to full-length human TR-2, suggestingthat amino acids 17-85 of the human sequence are important for binding.Overall, one or more of the amino acids in the region of 1-85 of thehuman sequence (SEQ ID NO: 96; ALITQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSG EVELSPCTTTRNTVCQ)are involved in epitope binding. Similarly, replacement of various humansequences in the region of amino acids 1-85 with the corresponding mousesequence significantly attenuates antibody binding (see FIG. 27),further confirming that one or more amino acids in that region areinvolved in epitope binding.

1-113. (canceled)
 114. An isolated antibody comprising a heavy chain anda light chain, wherein the heavy chain comprises: a) CDR1, CDR2, andCDR3 of SEQ ID NO: 2; b) CDR1, CDR2, and CDR3 of SEQ ID NO: 4; c) CDR1,CDR2, and CDR3 of SEQ ID NO: 6; d) CDR1, CDR2, and CDR3 of SEQ ID NO: 8;e) CDR1, CDR2, and CDR3 of SEQ ID NO: 10; f) CDR1, CDR2, and CDR3 of SEQID NO: 12; g) CDR1, CDR2, and CDR3 of SEQ ID NO: 14; h) CDR1, CDR2, andCDR3 of SEQ ID NO: 16; i) CDR1, CDR2, and CDR3 of SEQ ID NO: 18; j)CDR1, CDR2, and CDR3 of SEQ ID NO: 20; k) CDR1, CDR2, and CDR3 of SEQ IDNO: 22; l) CDR1, CDR2, and CDR3 of SEQ ID NO: 24; m) CDR1, CDR2, andCDR3 of SEQ ID NO: 26; n) CDR1, CDR2, and CDR3 of SEQ ID NO: 28; o)CDR1, CDR2, and CDR3 of SEQ ID NO: 30; p) CDR1, CDR2, and CDR3 of SEQ IDNO: 32; or q) CDR1, CDR2, and CDR3 of SEQ ID NO: 34; and wherein theantibody binds TRAIL receptor-2 (TR-2).
 115. The isolated antibody ofclaim 114, wherein the heavy chain comprises an amino acid sequence ofany of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, and
 34. 116. The isolated antibody of claim 114, wherein theantibody is a fully human antibody.
 117. The isolated antibody of claim114, wherein the antibody is linked to polyethylene glycol.
 118. Theisolated antibody of claim 114, wherein the antibody is glycosylated.119. An isolated antibody comprising a heavy chain and a light chain,wherein the light chain comprises: a) CDR1, CDR2, and CDR3 of SEQ ID NO:36; b) CDR1, CDR2, and CDR3 of SEQ ID NO: 38; c) CDR1, CDR2, and CDR3 ofSEQ ID NO: 40; d) CDR1, CDR2, and CDR3 of SEQ ID NO: 42; e) CDR1, CDR2,and CDR3 of SEQ ID NO: 44; f) CDR1, CDR2, and CDR3 of SEQ ID NO: 46; g)CDR1, CDR2, and CDR3 of SEQ ID NO: 48; h) CDR1, CDR2, and CDR3 of SEQ IDNO: 50; i) CDR1, CDR2, and CDR3 of SEQ ID NO: 52; j) CDR1, CDR2, andCDR3 of SEQ ID NO: 54; k) CDR1, CDR2, and CDR3 of SEQ ID NO: 56; l)CDR1, CDR2, and CDR3 of SEQ ID NO: 58; m) CDR1, CDR2, and CDR3 of SEQ IDNO: 60; n) CDR1, CDR2, and CDR3 of SEQ ID NO: 62; o) CDR1, CDR2, andCDR3 of SEQ ID NO: 64; p) CDR1, CDR2, and CDR3 of SEQ ID NO: 66; or q)CDR1, CDR2, and CDR3 of SEQ ID NO: 68; and wherein the antibody bindsTRAIL receptor-2 (TR-2).
 120. The isolated antibody of claim 119,wherein the light chain comprises an amino acid sequence of any of SEQID NOs: 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,and
 68. 121. The isolated antibody of claim 119, wherein the antibody isa fully human antibody.
 122. The isolated antibody of claim 119, whereinthe antibody is linked to polyethylene glycol.
 123. The isolatedantibody of claim 119, wherein the antibody is glycosylated.
 124. Anisolated antibody that specifically binds to an epitope that isspecifically bound by at least one antibody selected from: Ab A, Ab B,Ab C, Ab D, Ab E, Ab F, Ab G, Ab H, Ab I, Ab J, Ab K, Ab L, Ab M, Ab N,Ab O, Ab P, and Ab Q, wherein the antibody induces apoptosis in at leastone cell line selected from COLO 205 cells, MDA-231 cells, WM35 cells,and WM793 cells.
 125. The isolated antibody of claim 124, wherein theantibody binds to an epitope that is specifically bound by Ab O. 126.The isolated antibody of claim 124, wherein the antibody binds to anepitope within at least one amino acid sequence selected from SEQ ID NO:94, SEQ ID NO: 95, and SEQ ID NO:
 96. 127. The isolated antibody ofclaim 124, wherein the antibody binds to a polypeptide having the aminoacid sequence of SEQ ID NO:
 71. 128. A pharmaceutical compositioncomprising the isolated antibody of claims 114 and a pharmaceuticallyacceptable carrier.
 129. A pharmaceutical composition comprising theisolated antibody of claims 119 and a pharmaceutically acceptablecarrier.
 130. A pharmaceutical composition comprising the isolatedantibody of claims 124 and a pharmaceutically acceptable carrier.
 131. Amethod of treating cancer in a patient comprising administering atherapeutically effective amount of the antibody of claim
 114. 132. Amethod of treating cancer in a patient comprising administering atherapeutically effective amount of the antibody of claim
 119. 133. Amethod of treating cancer in a patient comprising administering atherapeutically effective amount of the antibody of claim 124.